PRACTICAL  HISTOLOGY 


AND 


THE  MICROSCOPE. 


A  COURSE 


PRACTICAL  HISTOLOGY: 


BEING    AN 


INTRODUCTION  TO  THE  USE  OF  THE 
MICROSCOPE. 


BY 
EDWARD  ALBERT  SCHAFER, 

ASSISTANT-PROFESSOR  OF  PHYSIOLOGY  IS  UNIVERSITY  COLLEGE,  LONDON. 


WITH  ILLUSTRATIONS  ON  WOOD. 


PHILADELPHIA: 

HENRY     C.     LEA. 

1877. 


S3 

£58 

6 


PHILADELPHIA ; 
COLLINS,     PRINTER 

7()."»  Jnyue  Street. 


PREFACE. 


THE  PURPOSE  of  this  work  is  to  afford  to  those 
engaged  in  the  practical  study  of  Histology,  plain 
and  intelligible  directions  for  the  suitable  prepara- 
tion of  the  animal  tissues ;  with  the  object  either 
of  immediate  study,  or  of  their  preservation  as  spe- 
cimens for  future  reference.  The  methods  recom- 
mended have  all  been  tested  by  experience. 

In  an  introductory  chapter  an  account  is  given 
of  the  several  parts  of  the  microscope,  and  the  pur- 
pose for  which  they  are  intended,  without  entering 
into  an  explanation  of  its  optical  construction  ;  and, 
in  an  Appendix,  instructions  wrill  be  found  for 
measuring  and  for  delineating  microscopic  objects. 

Throughout  the  book  descriptions  of  tissues  have 
been  purposely  avoided,  seeing  that  these  are  to  be 
found  in  systematic  works.  The  order  followed  is 
that  of  "Quain's  Anatomy,"  8th  Edition. 


Vlll  PREFACE. 

With  the  exception  of  Figure  12,  which  is  taken 
from  Dr.  Burdon  Sanderson's  "  Handbook  for  the 
Physiological  Laboratory,"  tLe  illustrations  have 
been  prepared  expressly  for  this  work ;  they  are 
from  the  pencil  of  Mr.  Collings. 

The  methods  employed  in  the  practical  study  of 
Embryology  have  been  omitted ;  they  will  be  found 
admirably  given  in  the  "  Elements  of  Embryology" 
of  Dr.  M.  Foster  and  Mr.  F.  M.  Balfour. 


UNIVERSITY  COLLEGE,  LONDON  : 

November,  1876. 


CONTENTS. 


INTRODUCTORY. 

PAGE 

Essential  parts  of  the  microscope — Accessory  parts — Appli- 
ances for  microscopic  work — General  directions  for  work  13 

CHAPTER   I. 

THE  BLOOD. 

Examination  of  human  blood — Methods  of  warming — Ac- 
tion of  reagents — Mode  of  applying  gases  or  vapors  to  a 
preparation — Frog's  blood — Phenomena  exhibited  by 
white  corpuscles — Action  of  reagents  on  frog's  blood — 
Mode  of  applying  electricity  to  a  preparation — Blood- 
crystals  25 

CHAPTER  II. 

THE  EPITHELIAL  TISSUES. 

Scaly  stratified  epithelium — Methods  of  separating  epithelial 
cells — Columnar  epithelium — Iodized  serum — Fixation  of 
cover-glass — Ciliated  epithelium — Ciliary  motion — Action 
of  reagents  on  ciliary  motion  .  .  .  .  .56 

CHAPTER  III. 

CONNECTIVE  TISSUE. 

Areolar  tissue — Fibres — Corpuscles — Action  of  acetic  acid — 
Method  of  localized  oedema — Ground  substance  and  cell 
spaces — Silver  method — Elastic  tissue — Fibrous  tissue — 
Tendon  cells  and  cell  spaces — Adipose  tissue  ...  70 


X  CONTENTS. 

CHAPTER  IV. 

CARTILAGE. 

PAGE 

Articular  cartilage — Action  of  water  on  cartilage  cell — Pre- 
servation of  cartilage  cell — Treatment  with  nitrate  of 
silver — Treatment  with  chloride  of  gold  (Cohnheim's 
method) — Warming  apparatus — Costal  cartilage — Fibro- 
cartilage — Staining  of  cartilage  matrix  with  logwood  .  89 

CHAPTER  V. 

BONE. 

Mode  of  grinding  sections — Effect  of  presence  of  air  in 
cavities  —  Modes  of  decalcifying  bone  —  Structure  of 
lamellae  —  Siiarpey's  fibres — Process  of  ossification  — 
Miller's  fluid— Marrow 100 

CHAPTER  VI. 

MUSCULAR  TISSUE. 

Involuntary  muscle — Isolation  of  cells — Arrangement  of 
cells  shown  by  silver  method — Voluntary  muscle  of  mam- 
mals— Action  of  acetic  acid — Frog's  muscle — Sarcolemma 
— Separation  of  fibre  into  disks  and  fibrils — Isolation  of 
fibres — Living  muscle  of  water-beetle — Contraction  of 
muscle — Examination  by  polarized  light — The  polarizing 
microscope  —  Sections  of  frozen  muscle  (Urban  Prit- 
chard's  method  of  freezing) — Ending  of  muscle  in  tendon  112 

CHAPTER  VII. 

NERVOUS  TISSUE. 

Medullated  nerves — Action  of  osmic  acid — Non-medullated 
nerves — Ensheathment  of  nerve — Silvered  nerves — Mode 
of  isolating  cells  of  spinal  cord — Ganglion  cells — Pacinian 
corpuscles — Motor  end-plates 126 


CONTENTS.  XI 

CHAPTER   VIII. 

THE  BLOODVESSELS. 

PAGE 

Epithelioid  lining  (silver  preparation) — Elastic  and  mus- 
cular layers — Method  of  preparing  sections — Embedding 
mass — Process  of  embedding — Mode  of  cutting  sections — 
Treatment  of  sections — Smaller  bloodvessels — Epithelioid 
lining — Muscular  structure — Study  of  circulation  in  frog's 
web — In  mesentery — In  mammals — In  lung  of  toad— In 
tongue — Inflammatory  changes — Mode  of  injecting  blood- 
vessels— Preparation  of  injection — Preparation  of  Berlin 
blue  —  Injecting  apparatus — Injection  of  an  animal  — 
Treatment  of  injected  ^issues 138 

CHAPTER  IX. 

LYMPHATICS  AND  SEROUS  MEMBRANES. 

Omentum  (silver  preparation)  —  Central  tendon  of  dia- 
phragm— Lymphatic  septum  of  frog — Injection  of  lym- 
phatics— Mercurial  apparatus — Method  of  puncture  in- 
jections— Injection  of  lymphatics  of  tendon — Treatment 
of  injected  preparation — Lymphatics  of  diaphragm  — 
Natural  injection — Synovial  membranes  —  Bloodvessels 
of  synovial  membranes — Haversian  fringes — Lymphatic 
glands 172 

CHAPTER  X. 

THE  SKIN,  HAIRS,  AND  NAILS. 

Mode  of  hardening — Method  of  double  staining — Tactile 
corpuscles — Bloodvessels  of  skin — Stirling's  digestion 
method — Hairs — Sections  of  hairs,  gum  method — Nails — 
Embedding  in  gum — Deferred  preparations — Bloodves- 
sels of  muscle — Sections  of  nerve  trunk — Lymph-spaces 
of  perineurium — Sections  of  ganglia  ....  185 

CHAPTER  XI. 

THE  HEART. 

t 

The  cardiac  pericardium — Muscular  substance — The  endo- 
cardium— Fibres  of  Purkinje— Lymphatics  of  heart  .  193 


Xll  CONTENTS. 

CHAPTER  XII. 

THE  LUNGS. 

PAGE 

The  pulmonary  pleura — The  costal  pleura — Mode  of  hard- 
ening lung — Embedding  in  cacao-butter — Kleinenberg's 
logwood — Epithelium  of  air-cells  shown  with  nitrate  of 
silver — Mode  of  injecting  lungs — Larynx  and  trachea — 
Thyroid  and  thymus 196 

CHAPTER  XIII. 

THE  MOUTH  AND  PHARYNX. 

Mucous  membrane  of  mouth— The  teeth — Sections  of  hard 
tooth — Sections  of  softened  tooth — foentinal  sheaths — 
Pulp  of  the  teeth — Decalcification  with  picric  acid — 
Teeth  in  situ — Development  of  the  teeth — Staining  with 
carmine — The  tongue — Mode  of  hardening  of  tongue — 
Conditions  of  mucous  glands — Bloodvessels  of  tongue — 
Palate  and  tonsils— .Salivary  glands — Different  condi- 
tions of  salivary  glands — Salivary  cells  treated  with 
osmic  acid 204 

CHAPTER  XIV. 

THE  (ESOPHAGUS  AND  STOMACH. 

Mode  of  hardening  gullet — Embedding  membranous  viscera, 
direction  of  sections — Vessels  of  gullet — The  stomach — 
Distension  with  chromic  acid  and  spirit  mixture — Gastric 
glands — Cardiac  and  pyloric  mucous  membranes — Stain- 
ing of  sections  with  carmine  and  aniline  blue — Cross- 
sections  of  glands: — Cells  of  glands — Bloodvessels  and 
lymphatics  of  stomach 210 

CHAPTER  XV. 

THE  SMALL  AND  LARGE  INTESTINE. 

Sections  of  small  intestine — Duodenum — Jejunum — Ileum 
— Process  of  fat  absorption — Bloodvessels  and  lymphatics 
of  small  intestine — Nerves  of  intestine  shown  by  gold 
method — Plexus  of  Meissner — Plexus  of  Aucrbach — 
Large  intestine  .  ..  217 


CONTENTS.  Xlll 

CHAPTER  XVI. 

THE  LIVER. 

PAGE 

Mode  of  hardening — Direction  of  sections — Portal  canals — 
Capillaries  of  lobules — Perivascular  lymphatics — Inter- 
cellular bile  channels — Mode  of  injecting  bloodvessels  of 
liver — Injection  of  bile-ducts  with  Berlin  blue — Injection 
of  lymphatics  of  liver — Study  of  liver  ceils — The  pancreas  223 

CHAPTER  XVII. 

THE  SPLEEN  AND  URINARY  ORGANS. 

Mode  of  hardening  spleen — Washing  out  of  bloodvessels — 
Injection  of  spleen — Teased  preparations  of  spleen — Reti- 
form  tissue — The  kidney — Mode  of  hardening  sections  of 
cortical  and  medullary  parts — Transverse  section  of  me- 
dullary part — Human  kidney — Injected  kidney — Isolation 
of  tubules — Fresh  kidney — Bowman's  capsule  and  base- 
ment membrane  of  tubules — Suprarenal  capsule — The 
ureters  and  bladder — Epithelium  cells  of  bladder  .  .  229 

CHAPTER  XVIII. 

THE  GENERATIVE  ORGANS. 

Erectile  tissue — Prostate  and  vesicula3  seminales — Scrotum, 
labia,  and  vagina — Human  uterus — Uterus  of  rabbit — 
Sections  of  ovary — Mammalian  ovum — Mode  of  finding — 
Artificial  impregnation  of  ovum — Testis — Injection  of 
lymphatics — Isolation  of  seminiferous  tubules — Basement 
membrane  of  tubules — Tunica  vaginalis — The  mammary 
glands  .  .  .  .  .  .  .  .  .  .  237 

CHAPTER   XIX. 

THE  CENTRAL  NERVOUS  SYSTEM. 

Preparation  with  bichromate  of  ammonia—  Use  of  microtome 
— Coloration  of  sections— Preparation  by  Sankey's  method     243 
B 


XIV  CONTENTS. 

CHAPTER  XX. 

THE  ORGANS  OF  THE  SENSES — THE  EYE. 

PAGE 

General  directions  for  hardening  in  osmic  acid  and  Miiller's 
fluid — The  eyelids — The  lachrymal  gland — The  sclerotic 
— Capsule  of  Tenon — Lamina  fusca — The  cornea — Sec- 
tions of  cornea — Epithelium  of  cornea— Substantiapropria 
of  cornea — Corneal  corpuscles— Frog's  cornea  stained 
with  gold— Prevention  of  creases  in  cornea — Rabbit's 
cornea  stained  with  gold — Mode  of  isolating  corpuscles 
with  potash — Nerves  of  rabbit's  cornea— Cell  spaces  of 
cornea  prepared  with  nitrate  of  silver — Injection  of  cell 
spaces — Nature  of  "  corneal  tubes" — Corneo-sclerotic 
junction  —  Ciliary  muscle  —  Lamina  suprachoroidea  — 
Gangliated  plexus  of  nerves — Layers  of  choroid — Mus- 
cular tissue  of  rabbit's  iris — Of  human  iris — The  retina — 
Hardening  in  Miiller's  fluid — In  osmic  acid — Staining  of 
— Embedding  in  cacao-butter — Teased  preparations  of 
retina — Preparation  with  chloral  hydrate — Fresh  retina — 
Hexagonal  pigment-cells — Ends  of  Miillerian  fibres  shown 
with  silver  nitrate — Retina  of  lower  vertebrata — The 
lens — Epithelium  of  lens  capsule — The  zonule  of  Zinn 
and  hyaloid  membrane  of  vitreous — Bloodvessels  of  the 
eye  .  . 246 

CHAPTER  XXI. 

THE  AUDITORY,  OLFACTORY,  AND  GUSTATORY  ORGANS. 

Semicircular  canals  of  skate— Cochlea  of  guinea  pig — Sec- 
tions of — Teased  preparations  of — Olfactory  mucous 
membrane  of  mammal — Olfactory  mucous  membrane  of 
amphibian — Taste-buds  of  rabbit 275 

APPENDIX. 

Method  of  measuring  an  object  under  the  microscope — De- 
termination of  magnifying  power  of  microscope — Mode  of 
drawing  microscopic  objects — Mode  of  counting  the  blood 
corpuscles — Microtomes  —  Microphotographic  apparatus 
— Employment  of  eosin  as  a  staining  fluid        .        .        .     285 

INDEX  297 


LIST  OP  ILLUSTRATIONS. 


ARRANGEMENT  OF  WORKING  TABLE      .        .     Frontispiece. 

FTG.  PAGE 

1.  Diagram  of  microscope 14 

2.  Dissecting  lens 18 

3.  Dissecting  microscope 18 

4.  Scissors,  forceps,  and  mounted  needle   .        .  21 

5.  Mode  of  letting  down  cover-glass 25 

6.  Simple  warm  stage 30 

7.  Simple  warming  apparatus  shown  in  actual  use    .        .  32 

8.  Osier's  phenomena 33 

9.  Warm  stage  with  thermometer  and  gas-regulator          .  35 

10.  Simple  moist- chamber 40 

11.  Gas-chamber 41 

12.  Clot  of  frog's  blood  in  capillary  tube     ....  46 

13.  Apparatus  for  passing  CO2  over  a  preparation       .        .  49 

14.  Slides  for  the  passage  of  electric  shocks  over  a  prepara- 

tion        50 

15.  Apparatus  for  passing  electric  shocks.    ....  51 

16.  Valve  of  mussel  showing  gills        .....  64 

17.  Pravaz  syringe        ........  75 

18.  Method  of  irrigation 90 

19.  Warming  apparatus  with  gas-regulator  for  gold  prepa- 

rations    95 

20.  Upper  part  of  gas-regulator    ......  96 

21.  Outline  of  paper  for  embedding  trough          .         .         .  143 

22.  Paper  folded  to  form  trough 144 

23.  Embedding  trough  of  capsule  metal,  on  cork,  with  tis- 

sue pinned  in  situ        .        .        .        .        ;        .         .144 


XVI  LIST    OF    ILLUSTRATIONS. 

FIG.  PAGK 

24.  Method  of  cutting  sections 146 

25.  Section-lifter 148 

26.  Cork  for  supporting  frog         ......  156 

27.  Structure  and  position  of  tongue  of  toad        .        .        .  159 

28.  Injecting  apparatus        .......  166 

29.  Canulas  for  injecting 168 

30.  Mercurial  pressure  apparatus  for  injecting     .         .         .  178 

31.  Perforated  steel  needle  for  puncture  injection        .        .  179 

32.  Tongue  of  rabbit    ........  282 

33.  Ocular  micrometer 286 

34.  Lines  of  stage  micrometer  viewed  with  ocular  micro- 

meter   287 

35.  Camera  lucida  for  drawing     ......  288 

36.  Apparatus  for  mixing  blood  and  for  counting  corpus- 

cles        289 

37.  Stirling's  microtome       .        .         .        .        .        .        .291 

38.  Hand  microtome    .        . 292 

39.  Rutherford's  freezing  microtome    .....  293 

40.  Microphotographic  apparatus 295 


PRACTICAL  HISTOLOGY. 


INTRODUCTORY. 

THE  practical  study  of  Histology  is  mainly  depen- 
dent upon  the  use  of  the  microscope.  The  microscope 
is  a  combination  of  lenses  arranged  for  the  purpose 
of  obtaining  and  viewing  a  magnified  image  of  any 
minute  object.  The  lenses  are  set  in  a  tube  of  vari- 
able length — the  tube  of  the  microscope  (Fig.  1,  £,  t') — 
and  this  is  itself  supported  in  a  vertical  position,  on 
a  firm,  metal  stand,  which  is  provided  with  an 
arrangement  by  which  the  tube  is  capable  of  being 
moved,  without  lateral  deviation,  in  a  perfectly 
straight,  up  and  down  direction.  This  arrange- 
ment is  termed  the  adjustment.  Its  purpose  is  to 
bring  the  microscope  into  that  position  with  regard 
to  the  object  in  which  the  latter  is  most  clearly 
seen.  The  object  is  then  said  to  be  in  focus. 

Two  adjustments  are  commonly  provided:  one — 
the  coarse  adjustment  (adj) — serves  to  bring  the  lenses 
roughly  into  the  focal  position,  and  is  either  a  tele- 
scopic joint  or  a  rack  and  pinion  movement;  the 
other — the  fine  adjustment  (adjf] — is  a  fine  screw,  and 
by  its  means  the  focus  may  be  obtained  with  com- 
plete exactness  even  when  the  highest  magnifying 
powers  are  employed.  The  stand  is  further  pro- 
vided with  a 'rigidly  connected,  horizontal  table  or 
stage  (st),  upon  which  the  object  is  placed,  and  which 
projects  below  the  tube  and  is  provided  with  a  cir- 
cular aperture  to  admit  light  from  below  to  the 
object,  capable  of  being  varied  by  means  of  a  dia- 
phragm (d)  furnished  with  holes  of  different  sizes. 
2 


14 


PRACTICAL    HISTOLOGY. 


Diagram  of  microscope. 

/,  tube  seen  in  section  ;  t',  sliding  part  of  tube  ;  oc,  ocular,  with  <?,  eye  glass,  and 
/.  field-glass  ;  obf,  objective ;  adj,  coarse,  and  «i.d/',  fine  adjustment;  ,fi,  sta^e; 
with  d,  diaphragm;  ni,  mirror;  ft,  foot  of  microscope. 

The  object  i* indicated  l.y  a  small  arrow  j  nst  above  the  aperture  in  the  diaphragm  ; 
the  magnified  image  by  the  larger  arrow  in  the  middle  of  the  ocular. 


THE    MICROSCOPE.  15 

Diffused  daylight  is,  if  possible,  employed,  and  is 
reflected,  by  means  of  a  movable  mirror  (m)  below 
the  stage,  up  through  the  object  and  through  the 
tube  of  the  microscope  to  the  eye  of  the  observer. 
This  is  termed  viewing  an  object  by  transmitted 
light.  Occasionally,  especially  when  comparatively 
large  and  opaque  objects  and  low  magnifying  powers 
are  to  be  employed,  the  former  are  viewed  by  the 
light  which  is  reflected  from  their  surface,  whilst 
that  from  the  mirror  is  cut  off.  In  order  in  such 
cases  to  concentrate  as  much  light  as  possible  upon 
the  object,  a  bull's-eye  condenser  is  employed.  It  is 
only  in  viewing  such  preparations  that  the  binocular 
microscope  offers  any  material  advantage  inhistology. 
The  lenses  form  the  essential  part  of  the  micro- 
scope, and  are  so  arranged  that  one  set  of  them, 
which  is  placed  at  the  lower  end  of  the  tube,  pro- 
duces a  magnified,  inverted  image  of  the  object  at 
the  upper  part.  The  image  thus  produced  is  viewed, 
and  at  the  same  time  still  further  magnified,  by  a 
lens  at  the  top  of  the  microscope  tube.  This  lens 
(e\  placed  close  to  the  eye  of  the  observer,  is  fixed 
in  the  eye-piece  or  ocular ;  but  this  part  includes  an- 
other glass  (the  field-glass,/)  situate  below  the  first 
and  fixed  in  one  piece  with  it,  having  for  its  object 
the  collection  of  the  more  divergent  rays  transmitted 
by  the  lower  set  of  lenses,  and  serving  also  to  lessen 
or  obviate  any  chromatic  aberration  which  might 
otherwise  be  produced.  The  whole  eye-piece  (oc) 
thus  composed  is  made  to  slip  in  at  the  superior 
aperture  of  the  microscope  tube.  The  lower  set  of 
lenses  form  an  achromatic  combination  which,  from 
its  situation  near  the  object,  is  termed  the  object-glass 
or  objective,  and  it  is  upon  the  perfection  of  its  con- 
struction that  the  useful  ness  of  the  microscope  mainly 
depends.  It  is  seldom  necessary  to  have  more  than 
one  ocular  of  medium  strength  in  use,  but  at  least 
two  objectives  of  different  magnifying  powers  are 
essential  for  histological  work.  One  of  these,  which 
in  subsequent  pages  will  be  spoken  of  as  the  low  power, 


16  PRACTICAL    HISTOLOGY. 

should,  when  used  with  the  ordinary  eye-piece,  give 
an  apparent  linear  enlargement  of  about  75  (75  dia- 
meters); that  is  to  say,  when  a  line,  the  length  of 
which  is  known  (say  y-J-Q-  of  .an  inch),  is  observed 
through  this  combination,  it  should  appear  seventy- 
five  times  as  long  as  it  really  is  (f  inch*  therefore). 
The  other,  to  be  mentioned  as  the  high  power,  should 
give  an  apparent  linear  magnification  of  from  300 
to  400  times.  These  two  glasses  amply  suffice  for 
all  ordinary  histological  studies;  but  for  certain  spe- 
cial subjects  it  is  advantageous  to  obtain  the  use  of 
a  more  powerful  combination;  one  that  will  magnify 
1000  diameters  or  more. 

Glasses  of  this  high  magnifying  power  are  usually  of 
the  kind  known  as  "immersion-objectives,"  so-called  be- 
cause they  are  made  so  as  to  be  used  with  a  stratum  of 
water  between  the  specimen  and  the  lower  lens  of  the 
objective.  The  water  (distilled)  is  applied  to  this  lens 
with  a  splinter  of  wood  or  from  a  pipette  before  the  objec- 
tive is  screwed  on  to  the  tube  of  the  microscope ;  the  tube 
is  then  lowered  by  means  of  the  coarse  adjustment  until 
the  drop  of  water  comes  in  contact  with  the  cover-glass, 
after  which  the  focus  is  obtained  by  cautiously  lowering 
it  further,  at  first  with  the  coarse,  and  then  with  the  fine 
adjustment. 

These  comprise  the  essential  requirements  of  a 
microscope,  but  larger  instruments  are  often  pro- 
vided with  certain  adjuncts  which  render  them  in  a 
measure  more  complete.  Thus  the  stand  may  be 
hinged  so  that  the  stage  and  tube  can  be  tilted 
somewhat  out  of  the  perpendicular  to  allow  of  better 
adaptation  to  the  position  of  the  observer.  But  if 
the  microscope  tube  is  not  inconveniently  high,  it  is 
almost  as  comfortable  to  work  without  inclining  the 
instrument.  Moreover,  very  many  preparations,  most 
of  those,  for  instance,  which  have  to  be  examined  in 
fluid,  will  not  admit  of  inclination.  A  camera  lucida 
(one  which  does  not  necessitate  the  tilting  of  the 
microscope)  is  useful  for  obtaining  an  exact  sketch 
of  the  outlines  of  an  object. 


THE    DISSECTING    MICROSCOPE.  17 

A  polarization  apparatus  is  occasionally  employed 
in  investigating  the  optical  properties  of  the  sub- 
stances which  compose  the  tissues.  (It  is  also  of  use 
in  helping  to  determine  the  nature  of  crystalline 
deposits  in  urine  and  other  fluids.)  In  connection 
with  the  employment  of  this  the  stage-plate  and 
tuhe  of  the  microscope  are,  in  the  best  instruments, 
made  capable  of  rotating  on  a  vertical  axis.  In 
smaller  instruments  this  movement  is,  as  a  rule, 
not  provided  for,  and  indeed,  although  convenient, 
is  by  no  means  essential. 

The  mechanical  stage  movement,  which  is  so  often 
fitted  to  microscopes  of  English  construction,  and 
other  appliances  which  tend  to  mar  the  perfect  sim- 
plicity of  the  instrument  when  in  ordinary  working 
trim,  serve  rather  to  detract  from  its  usefulness  for 
purposes  of  histology. 

Attention  to  the  foregoing  points,  of  which  per- 
haps perfect  steadiness  and  rigidity  of  the  stand  and 
stage  is  the  most  important  to  insist  upon,  will  aid 
the  student  in  selecting  a  useful  microscope  so  far  as 
the  body  of  the  instrument  is  concerned.  The  excel- 
lence of  the  objectives  can  only  be  competently  j  udged 
of  by  one  who  is  already  somewhat  conversant  with 
the  use  of  the  microscope.  Preparations  of  some  of 
the  tissues  or  fluids  of  the  body  (connective  tissue, 
blood,  salivary  corpuscles)  form  the  best  test  objects 
for  the  high  powers  of  an  instrument  that  is  to  be 
employed  in  histological  studies. 

Jn  addition  to  the  ordinary  instrument  (which  is 
generally  distinguished  as  the  "  compound"  micro- 
scope), it  will  be  found  very  convenient  to  have  a 
smaller  microscope  of  some  sort ;  so  as  to  be  better 
able  to  follow  the  needles  or  other  instruments  when 
the  operator  is  engaged  in  the  separation  and  manip- 
ulation of  minute  objects.  An  instrument  wrhich  is 
used  for  this  purpose  is  termed  a  dissecting  or  pre- 
paring microscope.  Any  simple  lens  which  is  mount- 
ed on  a  stand  will  serve  (Fig.  2),  and  even  the  hull's- 

eve  condenser,  which  is  generally  furnished  with  the 

c  o* 


18 


PRACTICAL    HISTOLOGY. 
Fig.  2. 


Simple  form  of  dissecting  microscope,  provided  with  three  lenses  of 
different  magnifying  power. 

Fig.  3. 


ACCESSORY    APPARATUS.  19 

microscope,  may  be  employed  as  a  lens  if  the  marginal 
part  is  covered  by  a  black-paper  diaphragm.  A  very 
convenient  form  of  dissecting  microscope  is  shown  in 
Fig.  3.  This  consists  of  a  small  compound  micro- 
scope furnished  with 'an  arrangement  for  reversing 
the  image,  so  that  the  object  appears  in  its  natural 
position  and  not  inverted.  It  is  provided  also  with 
a  prism  for  reflecting  the  light  in  a  direction  conve- 
nient for  the  eye,  and  is  placed  on  a  wooden  stand 
so  constructed  as  to  afford  support  to  the  arms  of  the 
operator.  The  ordinary  low  power  objectives  may 
be  used  with  it. 

Besides  the  microscope,  the  student  who  is  com- 
mencing the  practical  study  of  histology  will  find  it 
necessary  to  be  provided  with  the  following  simple 
instruments  and  appliances : — 

Glass-slides  and  cover-glasses  (see  Fig.  5). — The  mi- 
croscopic slides  are  oblong  slips  of  glass  upon  which 
the  object  is  placed.  In  this  country  they  are  always 
cut  of  the  uniform  and  convenient  size  of  three  inches 
by  one  inch.  The  glass  should  be  quite  free  from 
flaws  and  specks.  The  cover-glasses  can  hardly  be 
too  thin.  It  is  true  that  when  very  delicate  they  are 
likely  to  be  broken  in  the  cleaning  unless  great  care 
is  exercised ;  but  then,  on  the  other  hand,  many  high 
power  objectives  require  to  be  focussed  so  close  to  the 
object  that  a  thick  cover-glass  cannot  be  used  with- 
out risk  of  crushing  the  tissue,  and  perhaps  of  scratch- 
ing the  objective.  Asa  rule,  the  chief  difficulty  in 
cleaning  them  arises  from  the  fact  that  their  surfaces 
are  generally  covered  with  a  thin  film,  of  grease  or 
other  organic  matter  which  it  is  almost  impossible 
to  rub  off.  But  this  is  easily  got  rid  of  by  placing  a 
number  of  them  together  in  a  small  glass  beaker  and 
pouring  a  little  strong  nitric  acid  upon  them.  This 
quickly  destroys  every  trace  of  organic  matter.  The 
acid  is  then  poured  off,  and  the  cover-glasses  are 
thoroughly  rinsed  by  allowing  water  to  stream  over 
them  from  a  tap  for  two  or  three  minutes.  They 
are  then  to  be  kept  in  water  ready  for  use,  and  need 


20  PRACTICAL    HISTOLOGY. 

only  be  dried  when  wanted.  The  drying  is  effected 
by  a  thin  linen  cloth,  one  corner  of  which  is  laid  flat 
on  the  table,  and  the  cover-glass,  having  been  placed 
on  this,  is  gently  rubbed,  first  on  the  one  surface  and 
then  on  the  other,  with  another  corner  of  the  cloth. 
Square  cover-glasses  should  always  be  used  in  prefer- 
ence to  round  ones  The  most  convenient  size  for 
general  purposes  is  three-quarters  of  an  inch  square. 

Mounted  needles. — These  are  fine  sewing-needles 
mounted  in  a  wooden  handle,  with  about  a  quarter  of 
an  inch  of  their  point  projecting.  They  are  amongst 
the  most  useful  instruments  which  the  histologist 
possesses,  and  will  be  in  constant  requisition.  They 
must  always  be  kept  clean  and  sharp.  Xeedles  may 
also  be  mounted  in  crochet-needle  holders.  These 
have  the  advantage  that  fresh  needles  can  at  any  time 
be  readily  substituted. 

Scissors  and  forceps. — A  small  pair  of  scissors  with 
short,  straight  blades,  is  necessary.  Their  cutting 
edge  must  always  be  kept  very  sharp,  especially 
towards  the  point,  otherwise  they  are  worse  than 
useless.  A  small,  curved  pair  is  often  useful,  but 
these  are  much  more  difficult  to  keep  sharp.  At 
least  two  pairs  of  small  steel  forceps  are  requisite. 
The  blades  should  be  short,  broad  at  their  junction, 
so  as  not  to  admit  of  lateral  deviation,  and  towards 
the  end  gradually  tapering  to  a  blunt  point.  They 
are  slightly  roughened  at  the  end,  so  as  to  afford  a 
firmer  grasp.  The  long,  slender  forceps  which  are 
commonly  sold  as  microscopic  forceps  are  useless  for 
histological  purposes. 

Slips  of  white  bibulous  paper  should  always  be  at 
hand.  They  serve  both  -for  soaking  up  excess  of 
fluid  from  under  the  cover-glass  and  for  placing  the 
slide  upon,  when  preparing  a  tissue  that  has  been 
stained,  so  that  it  is  better  seen  than  it  would  be 
upon  a  black  surface.  On  the  other  hand,  it  is  better 
to  use  the  black  surface  for  working  upon  when  tis- 
sues are  unstained.  One  or  two  large,  shallow  glass 
dishes,  either  flat  (such  as  are  used  by  photographers) 


INSTRUMENTS. 


21 


Fig.  4. 


Scissors  and  forceps  for  histological  purposes.     Natural  size. 
7i,  end  of  mounted  ueedle. 


22  PRACTICAL    HISTOLOGY. 

or  watch-glass  shaped,  are  in  constant  requisition, 
since  very  many  of  the  manipulations  require  to  be 
performed  whilst  the  tissue  is  immersed  and  floated 
out  in  fluid;  while  for  applying  reagents  to  a  speci- 
men under  the  microscope  small  pipettes  are  ex- 
tremely convenient. 

Reagents. — Two  or  three  small  bottles  containing 
the  fluids  which  are  most  used  for  preserving  and 
mounting  preparations  should  be  always  on  the 
work-table.  Small  bottles  provided  with  a  cap  and 
with  a  piece  of  glass  tube  drawn  out  to  a  point  and 
standing  in  the  bottle  are  perhaps  the  best,  but  small 
corked  phials  with  a  piece  of  glass  tube  passed 
through  the  cork  will  answer  every  purpose.  One 
of  these  should  contain  a  mixture  of  glycerine  and 
water  (equal  parts  of  each);  another  a  solution  of 
Canada  balsam  in  chloroform  for  fixing  the  cover- 
glass  when  preparations  have  been  mounted  in  gly- 
cerine (this  should  have  a  small  brush  instead  of  a 
glass  tube) ;  and  another,  which  will  not  be  used  much 
at  first  but  will  afterwards  be  often  in  requisition, 
should  contain  dammar  varnish.  It  is  useful  also  to 
have  a  wash-bottle  of  distilled  water,  a  flask  of  salt 
solution,  a  solution  of  chloride  of  sodium,  containing 
1  part  of  the  salt  to  150  or  200  of  water,  and  a  small 
flask  of  recently  prepared  logwood  alum  stain  ing- 
solution.  This  is  made  by  taking  5  grammes  each 
of  powdered  alum  and  extract  of  logwood,  and  rub- 
bing them  up  thoroughly  together  in  a  mortar  with 
100  cc.  of  water.  The  mixture  is  covered  and  allowed 
to  stand  overnight,  it  is  then  filtered  into  a  bottle, 
and  a  lew  drops  of  a  solution  of  peroxide  of  hydrogen 
are  added.  The  solution  will  keep  two  or  three  weeks 
in  good  condition  for  staining,  but  it  must  always 
be  refiltered  immediately  before  being  used. 

Pipettes  may  be  readily  made  by  drawing  out  in 
the  flame  a  piece  of  soft  glass  tube  at  two  places 
close  to  one  another,  so  that  the  intermediate  part 
remains  as  the  bulb  of  the  pipette.  It  is  well  to 
make  a  number  at  a  time,  sealing  up  the  ends  in  the 


GENERAL    DIRECTIONS.  23 

flame  while  the  bulb  is  still  hot ;  they  are  thus  made 
absolutely  dust-tight  and  may  be  kept  always  ready 
for  use,  it  being  only  necessary  to  break  off  the 
sealed  ends  when  required  and  to  suck  the  reagent 
up  into  the  bulb.  A  pipette  should  always  be  re- 
jected after  it  has  once  been  used,  and  never  em- 
ployed for  another  reagent. 

For  keeping  preparations  that  have  been  per- 
manently mounted,  it  is  desirable  to  have  a  box  or 
cabinet  in  which  the  slides  can  be  put  away  in  a 
horizontal  position. 

General  directions  for  work.— Before  com- 
mencing see  that  the  table  is  in  order  and  clean, 
and  that  everything  is  at  hand  that  is  usually 
wanted  for  histological  work.  Especially  look  care- 
fully to  the  glasses  of  the  microscope  that  there  is 
no  dust  or  other  impurity  on  them.  If  any  glyce- 
rine or  Canada  balsam  should  have  found  its  way 
on  to  the  objective,  as  is  often  the  case  when  suffi- 
cient care  is  not  taken  in  placing  a  preparation 
upon  or  removing  it  from  the  stage,  they  are  to  be 
rubbed  off,  the  former  by  a  cloth  wetted  with  water, 
the  latter  with  a  little  spirit.  Before  beginning  to 
prepare  a  tissue  it  will  be  necessary  to  look  over  the 
description  of  the  mode  of  making  each  preparation, 
in  order  to  know  what  vessels  and  what  reagents  to 
get  together.  Otherwise  many  a  specimen  will  be 
spoiled  by  being  left  too  long  in  one  fluid  whilst  the 
one  to  which  it  should  be  transferred  is  being  got 
ready.  The  cover-glass  should  always  be  cleaned 
and  dried  before  commencing,  and  placed  ready  to 
hand  in  some  situation  where  it  is  not  likely  to  get 
broken.  It  is  well  always  to  put  the  cleaned  cover- 
glasses  in  the  same  place — say,  on  the  foot  of  the 
microscope — otherwise,  when  wanted  in  a  hurry  it 
is  often  difficult  to  find  them  at  once. 

Every  specimen  that  is  to  be  kept  must  be  dis- 
tinctively labelled  as  soon  as  made  ;  and  if  there  is 
anything  of  importance  to  remember  about  it  this 
must  be  at  once  entered  in"  the  notebook,  which  no 


24  PRACTICAL    HISTOLOGY. 

one  who  is  working  with  the  microscope  should  be 
without. 

Lastly,  the  student  should  never  trust  to  the 
transient  impression  of  form  or  structure  which  the 
mere  glance  at  a  microscopic  preparation  conveys, 
but  should  always,  whether  naturally  a  good 
draughtsman  or  not,  endeavor  to  perpetuate  the  im- 
pression so  obtained  by  a  careful  sketch  showing 
the  more  important  points  which  the  preparation 
illustrates.  The  rough  outlines  maybe  drawn  with 
a  camera  lucida  or  drawing  prism,  if  one  be  avail- 
able, but  even  without  such  an  instrument  a  little 
practice  soon  enables  a  sketch  to  be  produced  which 
gives  a  fairly  good  idea  of  the  appearances  seen,  and 
however  rough  it  may  be,  serves  materially  to  assist 
the  memory. 

For  the  most  part  it  is  also  desirable  to  note  the 
results  of  measurements  made  with  a  micrometer. 
The  use  of  this,  as  well  as  the  method  of  sketching 
an  object  by  aid  of  the  camera  lucida,  will  be 
described  in  the  Appendix  at  the  end  of  the  book. 


THE    BLOOD. 


25 


CHAPTER   I. 


THE  BLOOD. 

Preparation  1. — A  drop  of  blood  may  be  most 
conveniently  obtained  for  examination  from  the  fin- 
ger. It  is  generally  sufficient  to  give  the  end  of  the 
forefinger  of  the  left  hand  a  smart  prick  with  a  clean 
sewing-needle,  in  the  thin  part  of  the  skin  adjoining 
the  root  of  the  nail,  squeezing  firmly  with  the  right 
hand  above  the  point  pricked,  to  cause  a  drop  to 
exude.  If  necessary,  the  finger  may  first  be  con- 
gested by  tying  a  piece  of  string  tightly  round  it. 
As  soon  as  a  small  drop  of  blood  has  been  pressed 
out,  take  up  a  previously  cleaned  cover-glass  by  one 
edge  with  forceps,  let  the  drop  come  in  contact  with 
the  lower  surface  of  the  glass  near  the  opposite  ed«;e, 
so  that  a  little  adheres ;  and  then,  letting  this  edge 
come  first  in  contact  with  the  upper  surface  of  the 


Fig.  5. 


Glass  slide  and  cover-glass,  natural  size. 

The  figure  shows  the  mode  of  letting  down  the  cover-glass  (with  a  drop  of  blood 
on  its  under  surface)  gently  ou  to  the  middle  of  the  slide. 

slide  near   its  middle,  gradually  lower   the   other 
edge,  which  is  still  held  in  the  forceps,  on  to  the 
3 


26  PRACTICAL    HISTOLOGY. 

slide  (Fig.  5).  When  the  lower  blade  of  the  forceps 
nearly  touches  the  slide,  withdraw  the  instrument 
carefully,  so  that  the  cover-glass  may  now  rest  evenly 
upon  the  slide  by  its  whole  under  surface,  with  the 
blood  in  a  uniform  thin  layer  between.  It  is  impor- 
tant not  to  let  the  cover-glass  down  too  suddenly, 
for  if  dropped  carelessly  on  the  slide,  many  of  the 
corpuscles  will  be  broken  arid  destroyed.  When  the 
cover-glass  is  in  its  place,  there  ought  to  be  just 
enough  blood  entirely  to  fill  the  space  between  the 
two  glasses ;  but  it  is  better  to  have  too  little  than 
too  much.  It  might  be  supposed  that  the  delicate 
corpuscles  would  be  crushed  between  the  cover-glass 
and  slide,  but  they  are  for  the  most  part  protected 
from  this  by  the  buoying  up  of  the  cover-glass  by 
the  liquid  in  which  they  float. 

The  preparation  made,  it  is  to  be  at  once  trans- 
ferred to  the  stage  of  the  microscope,  and  examined 
with  a  power  of  about  BOO  diameters.  The  Held  of 
the  microscope  will  be  seen  crowded  with  corpus- 
cles floating  in  a  clear  liquid.  Probably  the  first 
thing  which  will  strike  the  beginner  is  the  very  faint 
color  which  the  so-called  red  corpuscles  present,  and 
these  will  also  most  likely  be  the  only  kind  of  cor- 
puscle that  he  will  at  first  be  able  to  distinguish. 
But  if  at  the  moment  'of  observation  there  happens- 
to  be  a  current  in  the  fluid — produced  either  acci- 
dentally by  a  shaking  of  the  room,  or  a  draught  of 
air,  or  purposely  by  gently  touching  the  cover-glass 
with  a  bristle — it  will  be  seen  that  while  most  of 
the  corpuscles  are  carried  along  by  the  current,  two 
or  three  remain  sticking  to  the  glass,  whilst  the 
others  are  carried  past  them;  and,  on  close  exami- 
nation, it  will  further  be  clear  that  these  are  of  a 
different  nature  from  the  rest,  being  entirely  devoid 
of  color,  and  of  a  pale  granular  appearance.  They 
are,  in  fact,  white  corpuscles,  and  once  seen,  will  be 
easily  recognized  again,  even  when  the  fluid  is  at  a 
standstill. 

Another  thing  that  will  be  made  manifest  by  tiny 


THE    RED    CORPUSCLES.  27 

motion  in  the  fluid  is  the  biconcave  discoid  shape 
of  the  red  corpuscles  ;  for  as  they  roll  over  it  will  be 
seen  that  their  outline  is  no  longer  circular  as  when 
lying  flat,  but,  on  the  contrary,  a  lateral  view  of  the 
disks  is  obtained,  and  the  flattening  and  incurvation 
of  the  surfaces  become  evident. 

When  the  motion  in  the  layer  of  blood,  in  what- 
ever way  it  may  have  been  produced,  is  subsiding,  it 
will  be  seen  that  whenever  one  corpuscle  comes  in 
contact  with  another  the  two  seem  to  be  in  some 
way  attracted  to  one  another,  so  as  to  adhere  closely 
by  their  opposed  surfaces;  and  other  corpuscles  com- 
ing in  the  same  way  in  contact  with  these  and  adher- 
ing, little  piles  or  rouleaux  are  thus  produced,  which 
form  by  their  junction  with  one  another  a  network, 
extending  throughout  almost  the  whole  of  the  pre- 
paration. In  the  cords  of  this  network  nearly  all 
the  red  corpuscles  are  involved,  and  now  for  the 
most  part  are  seen  edgeways;  but  in  other  parts  of 
the  preparation  where  the  layer  of  blood  is  very  thin 
— the  space  being  too  small  to  allow  the  corpuscles 
to  stand  edge  up,  and  to  combine  so  completely  to 
form  rouleaux — they  may  be  found  still  lying  flat 
and  distinct  from  one  another;  and  these  more  iso- 
lated corpuscles  may  now  be  subjected  to  careful 
examination.  Keeping  a  single  red  corpuscle  in 
view,  if  it  be  brought  exactly  into  focus — that  is  to 
say,  if  the  microscope  be  so  adjusted  that  the  con- 
tour of  the  corpuscle  is  as  distinct  as  possible — it  will 
be  observed,  with  the  power  (800  diameters)  which 
is  at  present  being  employed,  that  the  middle  part 
appears  slightly  darker  than  the  rim,  whereas  if,  by 
means  of  the  flue  adjustment,  the  objective  be  now 
brought  somewhat  nearer  (lower),  the  middle  part 
will  come  to  appear  lighter. 

The  cause  of  this  is  probably  to  be  found  in  the  xhape 
of  the  corpuscle,  the  middle  part  of  which  acts  upon  the 
light  like  a  very  weak  biconcave  lens,  refracting  the  rays 
of  light  which  nre  transmitted  through  it  slightly  out- 


28  PRACTICAL    HISTOLOGY. 

wards;  so  that  if  the  objective  is  at  a  certain  distance, 
all  of  the  rays  which  traverse  the  central  part  do  not 
reach  it,  some  of  them  being  deflected  too  much  to  the 
side  to  impinge  upon  the  lower  glass  of  the  objective. 
The  part  in  question,  therefore,  looks  a  little  dimmer  than 
the  somewhat  convex  marginal  part,  whereas  when  the 
objective  is  brought  nearer  all  the  rays  are  intercepted 
by  it;  and  the  middle  part. owing  to  its  greater  thinness, 
appears  lighter  than  the  rim.  If  a  very  high  power  ob- 
jective is  used,  the  middle  part  of  the  corpuscle  will  be  the 
lighter,  even  when  the  focus  is  rightly  adjusted,  for  in 
these  objectives  the  focal  length  is  generally  very  short, 
and  they  approach,  therefore,  near  enough  to  intercept 
the  outwardly  refracted  rays. 

With  the  exception  of  these  differences  of  shading 
(which  are  merely  dependent  upon  the  shape  of  the 
corpuscle),  the  red  corpuscles  present  a  perfectly 
homogeneous  appearance,  and  exhibit  in  the  fresh 
condition  no  tendency  to  separate  into  the  two  parts 
of  which,  as  the  study  of  the  action  of  reagents  will 
show,  they  in  reality  consist.  But  there  may  gener- 
ally be  noticed,  even  in  a  preparation  which  lias  been 
made  with  the  greatest  care,  a  red  corpuscle  here  and 
there  which  varies  from  the  prevailing  form,  having 
become  more  globular,  and  at  the  same  time  rather 
smaller  in  diameter;  some  of  these  retain  a  smooth 
contour,  whilst  others,  especially  those  near  the  edge 
of  the  preparation,  have  a  jagged  or  crenate  margin, 
as  if  set  with  little  projections,  and  these  may  also  be 
seen  on  the  surfaces  of  the  corpuscles  by  carefully  ad- 
justing the  microscope.  This  change  of  form,  which 
is  very  characteristic  of  the  mammalian  red  cor- 
puscles on  exposure,  seems  to  be  generally  caused  by 
a  shrinking  of  the  corpuscles,  induced  by  an  increase 
in  the  density  of  the  plasma  in  which  they  float;  it 
may  always  be  produced  by  adding  salt  to  blood. 

Turning  now  our  attention  to  the  white  corpus- 
cles, not  more  than  two  or  three  of  which  are  to  be 
seen  in  each  field  of  the  microscope  when  the  ordi- 
nary high  power  is  being  used,  and  which,  as  before 


THE    WHITE    CORPUSCLES.  29 

stated,  are  readily  distinguishable  from  the  red  cor- 
puscles by  their  want  of  color  and  their  pale,  granu- 
lar aspect,  we  usually  notice,  if  the  room  is  moder- 
ately cool,  and  provided  they  are  not  pressed  down 
by  the  cover-glass,  that  they  are  spheroidal  and  com- 
pletely motionless,  exhibiting  no  indications  of  vital- 
ity. Some  of  them  may  be  noticed  to  contain  a  small 
group  of  well-marked  granules,  much  coarser  than 
the  excessively  fine  granules  .which  pervade  the 
whole  substance;  and  in  conformity  with  this  it  is 
usual  to  describe  two  kinds  of  white  corpuscles — the 
finely  granular,  and  the  coarsely  granular — but  there 
would  not  seem  to  be  any  very  essential  difference 
between  the  two.  As  a  rule,  before  the  addition  of 
reagents,  no  nucleus  is  visible  in  either  variety, 
although,  as  will  be  afterwards  seen,  one  or  more  is 
always  present  in  each  ;  the  nuclei  are  delicate,  how- 
ever, and  readily  obscured  by  the  granules  of  the 
protoplasm.  If  the  room  is  tolerably  warm  it  may 
happen  that  the  white  corpuscles  no  longer  preserve 
their  rounded  outline,  but  that  from  one  side  or  an- 
other of  a  corpuscle  a  bud -like  process  extends  itself, 
to  be  again  retracted  into  the  body  of  the  corpuscle; 
spontaneous  changes  of  form  being  thus  effected 
which  resemble  those  which  are  presented  by  the 
common  fresh-water  amoeba,  and  are  hence  termed 
a amoeboid;"  but  in  a  cold  preparation  of  human 
blood,  like  that  under  examination,  these  movements 
are  seldom  extensive,  and  do  not  serve  to  effect  an 
actual  change  of  place  in  the  corpuscles  such  as  we 
shall  see  to  be  the  case  in  a  preparation  which  is 
artificially  warmed. 

Further,  there  may  generally  be  seen  in  a  prepa- 
ration of  blood  a  number  of  excessively  minute  pale 
granules,  which,  if  present  in  quantity,  may  be 
closely  grouped  together  here  and  there  into  masses 
or  "colonies"  of  various  shapes  and  sizes  (see  Fig.  8, 
«),  which  the  beginner  is  sometimes  apt  to  mistake 
for  white  blood-corpuscles.  But  the  objects  in  ques- 
tion have  a  much  fa  in  tor  aspect ;  and  although  under 


30  PRACTICAL    HISTOLOGY. 

certain  conditions  they — at  least,  their  constituent 
granules — may,  as  we  shall  presently  see,  exhibit 
indications  of  vitality,  yet  nothing  resemhling  in 
nature  the  amoeboid  movements  of  the  white  blood- 
corpuscles  is  ever  observed  in  them. 

Finally,  a  few  excessively  fine  and  delicate  threads 
of  fibrin  may  be  observed  stretching  in  different  di- 
rections across  the  field  of  the  microscope ;  but  to  *see 
these  distinctly  a  very  high  power  is  needed. 

Preparation  2. — In  order  properly  to  study  the 
vital  phenomena  which  are  displayed  by  the  white 
blood-corpuscles,  it  is  necessary,  in  the  case  of  man 
and  warm-blooded  animals,  to  maintain  the  drop  of 
blood  under  observation  at  or  near  the  temperature 
of  the  body.  For  this  purpose  we  employ  what  is 
known  as  a  warm  stage,  of  which  there  are  several 
forms  in  use.  The  simplest  consists  merely  of  an 
oblong  copper  plate  (Fig.  6),  two  inches  by  one  inch, 
from  one  side  of  which  a  rod  of  the  same  metal,  four 
or  five  inches  long,  projects.  This  plate  has  a  round 

Fig.  6. 


Simple  warm  stage,  with  preparation  upon  it  enclosed  between  two 
cover-glasses. 

aperture  in  the  middle,  half  an  inch  in  diameter, 
and  is  fastened  to  an  ordinary  slide  by  sealingwax. 
The  preparation  is  made  as  follows:  Take  first  a 
clean,  large-sized  (one  inch  square)  cover-glass,  which 
in  this  case  is  to  be  used  instead  of  a  slide,  and  on  it 
place  a  small  drop  of  salt  solution.  With  this  mix 


SIMPLE    WARM    STAGE.  31 

thoroughly  with  a  needle  about  an  equal  amount  of 
blood  obtained  from  the  finger,  as  in  Prep.  1,  and 
carefully  cover  the  mixed  fluid  with  another  cover- 
glass,  somewhat  smaller  than  the  first.  If  there  is 
now  not  enough  fluid  to  fill  the  space  between  the 
two  glasses,  add  a  little  more  salt  solution  at  one 
edge  of  the  smaller  cover-glass;  but  if,  on  the  other 
hand,  there  is  too  much,  soak  up  the  excess  with  a 
small  piece  of  blotting-paper.  A  very  small  camel- 
hair  pencil  which  has  been  dipped  in  olive-oil  is  now 
to  be  drawn  gently  along  each  edge  of  the  smaller 
glass;  this  will  prevent  evaporation  from  the  ed^es, 
which  would  otherwise  quickly  ensue  on  warming 
the  preparation.  The  dilution  of  the  blood  with 
salt  solution  prevents  in  great  measure  the  aggrega- 
tion of  the  red  corpuscles,  while  at  the  same  time  in 
no  way  interfering  with  the  movements  of  the  white 
ones;  moreover,  it  is  favorable  to  the  changes  which 
the  above-mentioned  masses  or  colonies  of  discoid 
particles  undergo,  if  any  such  happen  to  be  present. 
The  glass  slide  which  bears  the  copper  plate  having 
been  clamped  on  to  the  microscope  stage,  the  prepa- 
ration thus  made  is  placed  upon  the  copper,  and, 
having  been  brought  in  focus,  one  or  more  white 
corpuscles  are  selected  for  observation — a  high  mag- 
nifying power  being  used.  The  rod  is  now  heated 
near  its  end  by  a  small  spirit-lamp,  and  the  heat  is 
conducted  by  the  rod  to  the  copper  plate,  and  from 
this  is  transmitted  to  the  preparation,  close  to  which 
a  small  fragment  of  a  mixture  of  white  wax  and 
cacao-butter,  previously  made,  and  melting  at  about 
30°  C.,  is  to  be  placed  upon  the  copper  (Fig.  7).  The 
lamp  is  now  gradually  approached  along  the  rod 
until  it  arrives  at  a  spot  the  heat  transmitted  from 
which  is  just  sufficient  partially  to  melt  the  frag- 
ment, and  it  is  then  left  burning  at  that  spot,  for 
since  the  fat  employed  melts  at  about  the  tempera- 
ture of  the  body,  we  know  that  the  preparation  will 
now  be  also  warmed  nearly  to  the  same  point. 

It  will  be  seen  that  as  the  preparation  begins  to 


32 


PRACTICAL    HISTOLOGY. 


get  warm  the  white  corpuscles,  which  were  perhaps 
previously  rounded  and  inert,  begin  to  throw  out 
processes  and  exhibit  amoeboid  movements,  which 
become  more  and  more  marked  as  the  temperature 


Simple  warming  appirntus,  complete,  shown  in  operation. 

rises,  so  that  by  virtue  of  these  an  actual  change  of 
place  from  one  part  of  the  field  to  another  may  be 
effected.  It  is  well  in  making  this  observation  to 
select  a  single  corpuscle  and  to  sketch  its  outline  and 
that  of  its  more  immediate  surroundings  at  intervals 
of  half  a  minute.  As  the  corpuscles  become  spread 
out  in  creeping  along  the  glass,  one  or  more  nuclei 
may  sometimes  be  seen  indistinctly  in  them;  more 


OSLER'S  OBSERVATIONS.  33 

often,  perhaps,  clear  spaces  or  vacuoles  are  to  be  seen 
in  their  protoplasm. 

The  red  corpuscles  in  this  preparation  may  be  dis- 
regarded, for  they  show  no  trace  of  amoeboid  move- 
ment, but  only  become  slightly  eremite,  owing  to 
the  action  of  the  salt  solution.  The  slight  shaking 
movement  which  many  of  them  exhibit  is  the  mole- 
cular or  Brownian  movement  common  to  all  minute 
solid  particles  floating  in  a  liquid. 

The  changes  which  the  colonies  of  discoid  particles 
(see  p.  29)  undergo  under  the  present  conditions — i.e., 
dilution  of  the  blood  with  salt  solution  and  warmth — 
should  be  carefully  studied.  As  a  rule,  the  larger  the 
colony  the  more  actively  the  changes,  about  to  be  de- 
scribed, take  place.  If  there  are  none  to  be  seen  in  the 
blood  from  the  finger,  a  drop  may  be  obtained  from  some 
other  source  and  prepared  in  a  similar  way.  They  are 
common  in  the  blood  of  some  animals — e.g.,  the  rat — and 
are  generally  large  and  numerous  in  that  of  persons  who 
have  been  long  ill. 

The  masses  in  question  (Fig.  8,  a)  are  often  of  consider- 
able size,  many  times  larger  than  a  pale  blood-corpuscle. 
As  Osier  has  shown,  the  particles  which  compose  them 
are  free  in  the  circulating  blood,  and  only  run  together 
when  the  blood  is  drawn. 


Changes  seen  in  one  of  the  masses  or  colonies  of  discoid  particles  from  a 
drop  of  human  blood,  diluted  "with  salt  solution  and  warmed     After  Osier. 

About  half  an  hour  after  the  preparation  has  been  made 
the  masses  may  be  seen  to  have  no  longer  an  even,  toler- 
ably well-defined  contour,  but  to  be  bristling  with  exces- 
sive minute  filamentous  projections,  each  of  which  has 
grown  out  from  one  of  the  minute  discoid  particles  at  the 


34  PRACTICAL    HISTOLOGY. 

exterior  of  the  clump  (Fig.  8,  b) ;  and  as  the  filament  gets 
a  little  longer  and  projects  more  the  remains  of  the  disk 
may  often  be  seen  forming  an  enlargement  near  its  middle. 
Soon  some  of  the  projecting  filaments  begin  to  oscillate 
in  the  liquid,  and  at  length  break  away  altogether  from 
the  mass,  and  continuing  their  oscillatory  movement  in 
the  surrounding  fluid,  become  gradually  more  and  more 
removed  from  the  clump,  until  they  may  eventually  .even 
pass  altogether  out  of  the  field  of  the  microscope.  Mean- 
while other  of  the  disks  have  grown  out  into  filaments 
and  taken  the  place  of  the  liberated  superficial  ones,  and 
soon  these  in  their  turn  become  free  and  give  place  to 
others ;  so  that  in  this  way  the  previous  solid-looking 
mass  becomes  almost  entirely  broken  up  into  freely-mov- 
ing filamentous  particles  (Fig.  8,  c).  These  are  not  all 
of  the  shape  above  described,  but  present  various  forms, 
as  shown  in  the  accompanying  figure. 

The  above  observation  shows  that  the  discoid  parti- 
cles which  are  occasionally  found  in  theblood^and  which 
when  the  blood  is  drawn  tend  to  become  collected  into 
clumps,  are  to  be  regarded  as  centres  of  origin  from  which 
minute,  filamentous,  bacterium-like  organisms  may  under 
some  circumstances  be  developed ;  but  whether  this  change 
ever  occurs  in  the  living  body,  and  what  further  modifi- 
cation the  filaments  may,  under  conditions  more  favorable 
for  their  development,  undergo,  is  at  present  undeter- 
mined. 

After  the  observations  recorded  in  the  preceding 
paragraph  are  completed,  the  action  of  an  excess  of 
heat  may  be  observed;  but  it  is  better  to  use  for 
this  purpose  a  larger  apparatus,  in  which  the  degree 
of  heat  can  be  measured  by  a  thermometer.  Such  a 
one  is  shown  in  Fig.  9.  In  this  the  preparation  is 
placed  upon  the  brass  box  «,  which  rests  on  the  stage 
of  the  microscope,  and  is  pierced  in  the  centre  by  a 
tubular  aperture,  to  admit  light  to  the  object.  The 
box  is  connected  by  India-rubber  tubes  with  a  hollow 
metal  jacket, /,  and  the  whole  system  thus  consti- 
tuted is  completely  filled  with  water  previously 
boiled,  to  the  exclusion  of  air.  The  water  is  warmed 
at  q  l>v  n  small  gas- flame,  and  rising  through  the  tube 

t/        •/  ,~  » 


MAINTAINING    TEMPERATURE. 


35 


c  communicates  its  beat  to  the  box  «,  tbe  temperature 
of  which  is  measured  by  a  small  thermometer  6,  in- 
serted through  an  obliquely  placed  tube,  quite  into 
the  central  hole  and  immediately  under  the  prepara- 


Fig.  9. 


Apparatus  for  maintaining  a  constant  temperature  under  the  microscope. 

tion.  The  cooled  water  from  the  stage  descends  down 
the  tube  c',  to  pass  again  round  to  the  flame,  and  in 
this  way  the  water  constantly  circulates.  The  bulbed 
tube  d,  tilled  with  mercury,  serves  to  regulate  the  flow 
of  gas,  so  as  to  keep  the  temperature  constant  at  any 
desired  point.  This  is  effected  by  turning  the  steel 
screw  <?,  when  this  point,  whatever  it  may  be,  is 
reached,  so  as  to  raise  the  mercury  in  the  glass  tube, 
and  thus  almost  block  up  the  lower  end  of  a  small 
steel  or  o-lass  tube,  which  is  fixed  into  the  upper  end 
of  the  tube  d.  The  gas  passes  through  the  small  tube, 
and  then  above  the  mercury  and  between  the  two 
tubes  to  be  conducted  by  the  side  piece  h  to  the 
burner  below,  and  it  will  be  understood  that  if  the 
temperature  now  rose  higher  in  the  reservoir/,  which 
surrounds  the  mercury,  this  on  being  warmed  will 


36  PRACTICAL    HISTOLOGY. 

expand  and  tend  to  cut  off  more  of  the  gas  and  thus 
reduce  the  flame,  on  which  the  mercury  will  again 
contract,  and  the  flame  will  rise  in  consequence,  and 
so  on.  It  is  found  that  an  equilibrium  soon  becomes 
established,  and  the  temperature  of  the  water  and 
stage  remains  almost  absolutely  constant.  To  raise 
or  lower  the  temperature  all  that  is  required  is  to 
screw  out  or  in  the  screw  e.  The  small  included 
tube  is  pierced  with  a  minute  aperture,  to  allow  a 
constant  passage  of  gas,  so  as  to  prevent  the  flame 
from  being  extinguished  in  the  event  of  the  complete 
occlusion  by.  the  mercury  of  the  lower  end  of  the 
tube  in  question. 

By  employing  this  or  a  similar  apparatus  it  will 
be  found  that  up  to  and  a  little  beyond  the  normal 
temperature  of  the  blood  the  white  corpuscles  become 
more  active  in  their  movements,  but  on  gradually 
warming  the  preparation  still  more  a  point  is  soon 
reached  at  which  they  draw  in  their  processes,  be- 
come spherical,  and  show  no  longer  any  signs  of 
vitality — the  temperature  being  sufficient  to  kill  the 
corpuscles.  The  red  corpuscles  remain  unaltered 
until  a  temperature  of  about  55°  C.  is  reached,  when 
they  become  altered  in  shape,  and  globular  ;  eventu- 
ally their  coloring  matter  is  discharged  and  becomes 
dissolved  out  in  the  surrounding  serum. 

ACTION  OP  REAGENTS  UPON  THE  BLOOD. — The  red 
corpuscles  in  the  two  preceding  preparations  ap- 
peared, even  under  the  highest  power,  perfectly 
homogeneous  and  structureless,  but  it  can  be  shown, 
by  the  application  of  reagents  to  the  blood  under 
the  microscope,  that  they  in  reality  consist  of  two 
intimately  mingled  but  separable  parts — the  stroma 
(formed  of  various  chemical  compounds,  such  as 
cholesterin,  paraglobulin,  &c.),  which  is  colorless, 
and  gives  the  shape  to  the  whole  corpuscle  ;  and  the 
colored  part,  which  consists  chiefly  or  wholly  of  a  red 
crystallizable  substance,  haemoglobin.  The  mode  of 
application  of  reagents  is  as  follows :  A  drop  of 
blood  is  got  ready  as  in  the  first  preparation,  and 


ACTION    OF    REAGENTS    ON    THE    BLOOD.      37 

whilst  under  observation  a  small  drop  of  the  reagent 
(which  should  as  a  rule  be  freshly  prepared)  is  al- 
lowed to  come  into  contact  with  the  edge  of  the 
cover-glass.  Some  of  the  fluid  flows  under  this  and 
mixes  with  the  drop  of  blood  ;  the  current  produced 
by  it  at  first  drives  the  corpuscles  before  it,  but  they 
soon  become  stationary,  and  then  the  part  of  the 
preparation  should  be  selected  for  observation  where 
the  reagent  is  gradually  diffusing  itself  amongst  the 
corpuscles.  In  this  way  every  stage  in  its  action 
may  readily  be  studied. 

Preparation  3.  Action  of  water. — When  a 
drop  of  distilled  water  is  applied  in  the  manner 
above  described,  the  first  efleet  is  seen  to  be  that  the 
red  corpuscles  begin  to  lose  their  discoid  form,  first 
one  of  their  sides  becoming  bulged  out,  so  that  they 
are  cup-shaped,  and  then  the  other  side,  so  that  they 
are  now  completely  globular,  as  may  be  seen  when 
they  roll  over.  Meanwhile  the  haemoglobin  is  being 
dissolved  out  of  the  corpuscles  by  the  water,  so  that 
they  are  soon  quite  colorless  and  hardly  to  be  de- 
tected in  the  now  reddish  fluid  Some  seem  to  offer 
greater  resistance  to  the  action  of  the  water  (and 
indeed  of  most  reagents)  and  to  retain  their  coloring 
matter  longer  than  others. 

The  white  corpuscles  are  also  soon  affected.  They 
cease  their  amoaboid  movements,  and  begin  to  swell 
up  by  imbibition  of  fluid,  whilst  at  the  same  time 
with  a  high  power  the  granules  in  their  interior 
may  be  seen  to  exhibit  the  dancing  movement  which 
is  characteristic  of  minute  particles  floating  in 
liquid.  Often  the  corpuscles  present  clear  bulgings 
at  their  circumference,  or  their  substance  may  ap- 
pear to  burst  at  one  point  and  become  diffused  in 
the  water.  As  they  swell  and  become  clearer,  one, 
two,  or  more  small  round  nuclei  generally  come  into 
view,  and  soon  these  also  become  swollen,  and  with 
the  rest  of  the  corpuscles  eventually  disintegrate, 
nothing  being  left  but  a  few  granules. 

Water  is  thus  proved  to  have  a  characteristic  ac- 
4 


38  PKACTICAL    HISTOLOGY. 

tion  upon  the  protoplasmic  white  corpuscles  as  well 
as  upon  the  very  easily  alterable  red  disks,  and  this 
fact  must  be  borne  in  mind  in  investigating  the  ac- 
tion of  reagents  or  poisonous  substances,  both  upon 
the  blood-corpuscles  and  upon  the  tissues  generally. 
If  a  reagent  is  to  be  employed  in  weak  solution, 
therefore,  it  is  well  to  dissolve  it  either  in  salt  solu- 
tion or  in  fresh  serum1  instead  of  water.  Washing 
with  water  tissues  which  are  subsequently  to  be  sub- 
mitted to  microscopical  examination,  as  is  so  freely 
done  in  the  post-mortem  room,  is  for  a  similar  rea- 
son to  be  deprecated  ;  but  if  a  trace  of  bichromate  of 
potash  or  of  chromic  acid,  or  a  little  common  salt  be 
previously  added  to  the  water,  its  deleterious  effect 
upon  the  protoplasm  is  in  great  measure  obviated. 

Preparation  4.  Action  of  Acetic  Acid. — To 
investigate  the  action  of  dilute  acids,  it  is  best,  as 
just  explained,  to  mix  the  acid  with  salt  solution 
instead  of  water ;  1  part  of  glacial  acetic  acid  to  200 
of  salt  solution  is  an  appropriate  strength  for  the 
blood.  The  preparation  is  made  in  the  usual  way, 
and  the  drop  allowed  to  run  in  at  the  edge  of  the 
cover-glass.  The  action  of  the  weak  acid  upon  the 
red  corpuscles  is  seen  to  be  quite  like  that  of  water; 
they  are  first  rendered  globular  and  then  decolor- 
ized. Upon  the  white  corpuscles  it  has  a  somewhat 
different  action,  for  although  the  protoplasm  of  the 
corpuscle  becomes  partly  swollen  out  into  a  clear 
spheroid,  the  nuclei  are  not  swollen  by  the  reagent, 
but  are  brought  very  distinctly  into  view,  as  well  as 
the  nucleoli  within  them,  and  remain  usually  at  one 
side  of  the  corpuscle,  with  a  little  granular  matter 
precipitated  around  them. 

Preparation  5.  Action  of  Tannic  Acid. — The 
action  of  tannic  acid  upon  the  red  corpuscles  is  pe- 
culiar and  interesting.  Like  other  acids  it  tends  to 
cause  the  colored  part  of  the  red  corpuscle  to  become 

1  The  scrum  employed  must  be  from  the  blood  either  of  the 
same  animal  or  ot'uue  belonging  to  the  same  species. 


MOIST    CHAMBER.  39 

separated  from  the  stroma ;  but  as  the  colored  ma- 
terial is  exuding  it  becomes  coagulated  by  the  astrin- 
gent reagent,  and  in  place  of  being  dissolved  in  the 
surrounding  liquid,  as  after  the  action  of  acetic  acid, 
it  remains  attached  to  the  stroma  as  a  small,  bright, 
reddish  projection. 

In  the  first  part  of  the  reaction,  viz.,  the  render- 
ing the  corpuscles  globular,  it  acts  similarly  to  other 
weak  acids. 

The  most  convenient  strength  of  solution  to  use 
is  1  per  cent.;  it.  should  be  freshly  prepared.  It  is 
well,  moreover,  first  to  mix  the  drop  of  blood  upon 
which  it  is  desired  to  test  the  action  of  the  reagent 
with  an  equal  amount  of  salt  solution ;  otherwise 
the  tannic  acid  produces  such  a  dense  precipitate 
with  the  albumen  of  the  serum  that  the  view  of  the 
corpuscles  is  greatly  obscured,  and,  indeed,  the  re- 
agent with  difficulty  reaches  them. 

Preparation  6.  Action  of  Weak  Alkalies.— 
A  mixture  of  1  part  .of  caustic  potash  to  500  of  salt 
solution  may  be  used,  a  drop  being  added  to  the 
preparation  in  the  usual  way  (p.  37).  The  reaction 
takes  place  very  suddenly  ;  the  corpuscles,  both 
white  and  red,  swell  up  as  soon  as  the  reagent 
reaches  them,  appear  to  burst,  au.d  then  entirely  dis- 
appear. The  white  are  affected  by  a  weaker  solu- 
tion than  the  red.  This  is  apparent  from  the  fact 
that,  as  the  liquid  slowly  diffuses  and  mixes  with 
the  blood,  the  white  may  be  seen  to  become  destroyed 
in  parts  where  the  red  are  still  unaffected. 

Preparation  7.  Chloroform. — To  observe  the 
action  of  chloroform  vapor  upon  blood  a  moist 
chamber  is  used.  This  is  an  apparatus  for  keeping 
a  tissue  or  fluid  under  examination  in  its  naturally 
moist  condition,  whilst  at  the  same  time  allowing 
its  surface  to  be  exposed  to  air  or  to  any  desired  gas 
or  vapor.  The  simplest  form  of  moist  chamber  is 
made  out  of  a  small  piece  of  soft  putty  or  modelling- 
wax,  which  has  been  rolled  out  between  the  fingers 
into  a  round  cord  about  2  inches  long  and  J  inch 


40  PRACTICAL    HISTOLOGY. 

thick  ;  the  ends  of  the  cord  are  united  so  as  to  form 
a  ring,  and  this  is  placed  on  the  middle  of  a  clean 
glass  slide.  A  drop  of  water  is  put  into  the  centre 
of  the  ring  ;  this  is  for  the  sake  of  keeping  the  at- 
mosphere of  the  chamber  rnoist ;  but  the  object  may 
be  equally  well  effected  by  breathing  into  the  space 
as  it  is  being  covered  over.  The  object  is  prepared 
on  the  centre  of  a  clean  cover-glass,  which  is  then 
inverted  over  the  ring,  so  that  the  preparation  is 
dependent  into  the  chamber,  and,  whilst  freely 
exposed  to  the  air  in  this,  is  entirely  protected  from 
evaporation  and  may  be  readily  examined  through 
the  cover-glass,  to  the  under  surface  of  which  it 
remains  adherent  (Fig.  10). 

Fig.  10. 


To  investigate  the  action  of  chloroform  vapor  it 
is  necessary  to  have  some  means  of  passing  this  into 
the  moist  chamber,  whilst  the  drop  of  blood  is  under 
observation.  For  this  purpose  a  slide  is  employed 
(Fig.  11),  to  which  a  piece  of  small  glass  tubing  has 
previously  been  fixed  by  means  of  sealing-wax,  and 
the  ring  of  putty  is  so  placed  as  to  include  the  end 
of  this,  a  small  interval  being  left  at  the  side  of  the 
tube  to  afford  an  exit  for  the  vapor.  The  slide  is 
then  clamped  on  to  the  microscope  stage  (as  in  Fig. 


MOIST    CHAMBER.  41 

13  6'),  and  the  glass  tube  is  connected  by  India-rubber 
tubing  to  a  bottle  containing  a  few  drops  of  chloro- 
form and  furnished  with  a  second  tube,  through 
which  air  can  be  blown. 


Fig. 


Chamber  for  passing  a  gas  or  vapor  over  a  preparation  under  the 
microscope. 

Before  the  cover-glass  is  superposed  the  blood 
should  first  have  been  spread  out  upon  it  into  a  thin 
layer,  so  that  the  chloroform  vapor  may  readily  act 
upon  all  parts. 

Everything  being  thus  prepared,  and  some  of  the 
blood  corpuscles  having  bean  brought  clearly  under 
observation,  air  is  blown  gently  into  the  bottle,  and 
passing  through  it  becomes  charged  with  the  vapor 
of  chloroform,  which  is  conveyed  by  the  tube  into 
the  moist  chamber,  where  it  acts  upon  the  layer  of 
blood  which  is  on  the  under  surface  of  the  cover- 
glass.  After  a  short  time  it  will  be  seen  that  the 
red  corpuscles,  as  under  the  action  of  water  and 
dilute  acids,  at  first  become  globular,  and  subse- 
quently their  haemoglobin  becomes  dissolved  and 
discharged  out  in  the  serum.  It  will  further  be  ob- 
served, both  in  this  and  in  the  other  preparations  in 
which  this  change  has  taken  place  in  the  red  cor- 
puscles, that  to  the  naked  eye  the  blood  has  changed 
from  scarlet  to  lake,  and  that  whereas  when  the  cor- 
puscles were  intact  even  a  thin  layer  of  blood  pre- 
sented a  somewhat  opaque  appearance,  it  is  now 

4* 


42  PRACTICAL    HISTOLOGY. 

completely  transparent.     Hence  we  may  infer  that 
the  opacity  of  the  unaltered  blood  is  due  to  the 
presence  of  the  red  particles. 
Preparation  8.   Examination  of  Frog's  Blood. 

— The  simplest  way  to  obtain  frog's  blood  for  exami- 
nation is  to  cut  off  the  tip  of  one  of  the  digits,  having 
previously  wiped  it  dry  with  a  cloth,  and  to  collect 
upon  a  clean  cover-glass  the  small  drop  of  reddish 
fluid  which  exudes.  The  glass  is  then  inverted  upon 
a  slide  and  the  drop  is  examined.  The  blood  so 
obtained  is  mixed  with  lymph,  and  the  corpuscles 
are  consequently  less  crowded  and  better  adapted 
for  observation  than  when  the  blood  is  undiluted. 
To  procure  blood  unmixed  with  lymph  the  animal 
should  be  pithed,  and  laid  upon  its  back.  The  heart 
is  then  exposed  and  snipped  with  scissors,  and  a  small 
drop  of  the  blood  which  exudes  is  taken  up  with  a 
glass  rod,  transferred  to  a  slide  and  covered.  But 
before  placing  the  cover-glass  down  in  the  usual  way 
a  small  length  (J  inch)  of  a  delicate  hair  should  be 
placed  in  the  drop,  so  that  when  the  cover-glass  set- 
tles down,  the  corpuscles,  here  comparatively  large, 
may  not  be  crushed  by  its  weight. 

When  the  preparation  obtained  from  either  of 
these  sources  is  examined,  it  will  at  once  be  seen 
that  the  colored  corpuscles  are  larger  and  fewer  in 
number  than  in  human  blood,  and  do  not  tend  to 
form  rouleaux ;  that  they  have  an  elliptical  outline 
when  lying  flat,  but  when  seen  edgeways  look  quite 
narrow  and  pointed  at  the  ends,  with  a  slight  and 
gradual  bulging  at  either  side;  so  that,  although 
disk-shaped,  like  the  mammalian  blood-corpuscles, 
so  far  from  being  biconcave,  they  are  biconvex.  The 
bulging  is  due  to  the  presence  in  the  middle  of  the 
corpuscle  of  another  part  besides  the  stroma  and  the 
colored  substance  found  in  the  mammalian  disk. 
This,  the  so-called  nucleus,  can  readily  be  made  out 
in  most  of  the  corpuscles  as  a  somewhat  elongated, 
colorless,  and  slightly  granular  elliptical  body,  about 
a  third  the  length  of  the  corpuscle,  and  often  lying 


FEEDING    OF    WHITE    CORPUSCLES.  43 

not  quite  in  the  middle  but  somewhat  eccentrically. 
Occasionally  the  nucleus  is  seen  to  be  round;  but 
this  is  an  accidental  change,  and  may  be  brought 
about  by  mechanical  injury.  Indeed,  if  the  precau- 
tions above  recommended  for  avoiding  pressure  are 
not  taken,  a  large  number  of  the  corpuscles  become 
injured,  so  much  so  as  even  to  be  ruptured  and  de- 
stroyed altogether,  in  which  case  the  rounded  nuclei 
are  liberated,  and  may  be  mistaken  by  the  beginner 
for  pale  blood-corpuscles. 

With  regard  to  the  white  corpuscles,  it  will  be 
observed  that  they  are  more  numerous  and  larger 
than  in  human  blood.  Moreover,  they  soon  begin, 
even  in  the  cold,  to  exhibit  very  distinct  amoeboid 
movements;  and,  on  account  of  the  greater  size  of 
the  corpuscles,  both  these  and  the  other  phenomena 
exhibited  by  them  are  much  more  striking,  and 
tbese  corpuscles  are  therefore  much  better  suited  for 
observation  than  those  of  mammals,  which  in  other 
respects  they  closely  resemble.  The  distinction  be- 
tween the  finely  and  the  coarsely-granular  corpuscle 
is  met  with  again  here;  but  there  is  also  sometimes 
seen  a  third  kind  of  corpuscle,  fusiform  or  oat-shaped 
and  devoid  of  amoeboid  properties.  It  has  been  sug- 
gested that  these  are  cells  which  have  belonged  to 
and  have  become  detached  from  the  lining  membrane 
of  the  bloodvessels. 

Preparation  9.  Feeding  of  the  White  Corpus- 
cles.— The  white  corpuscles  of  the  blood  exhibit  a 
strong  tendency  to  take  into  their  interior  any  small 
particles  of  insoluble  materials  which  may  happen  to 
be  in  their  neighborhood.  This  tendency  they  have 
in  common  with  all  amoeboid  organisms,  and  it  is 
nowhere  better  seen  than  in  the  case  of  the  amoeba 
itself.  Because  of  their  greater  size,  number,  and 
activity,  it  is  better  to  take  the  frog's  corpuscles  for 
this  experiment  rather  than  those  of  mammalia. 

To  obtain  the  white  corpuscles  in  quantity,  and 
comparatively  free  from  colored  ones,  take  a  capil- 
lary tube  (like  those  used  for  preserving  vaccine 


44  PRACTICAL    HISTOLOGY. 

lymph  in,  which  can  be  easily  made  by  heating  a 
piece  of  glass  tube  red-hot  in  the  blowpipe-flame, 
and  then  removing  and  drawing  it  out  before  it  has 
time  to  cool),  and  allow  it  to  till  with  blood  from 
the  heart,  exposed  as  above  directed.  Now  lay  the 
tube  aside  for  an  hour,  and  meanwhile  the  particles 
with  which  the  white  corpuscles  are  to  be  fed  may 
be  prepared.  This  is  done  in  the  following  way : 
A  few  drops  of  salt  solution  are  poured  into  a  watch- 
glass,  and  a  cake  of  Indian  ink  is  rubbed  in  it  until 
when  thoroughly  mixed  up  the  fluid  has  acquired 
a  faint  grayish  color.  When  left  to  itself  fora  time 
the  coarser  particles  subside,  whereas  the  finer  ones 
remain  longer  suspended.  At  the  expiration  of  the 
hour  the  blood  is  taken  and  its  contents  are  blown 
out  upon  a  glass  slide,  the  clot  which  has  formed 
within  the  tube  being  removed,  leaving  merely  the 
exuded  serum.  This  contains  a  few  red  blood-cor- 
puscles and  a  large  number  of  white  ones,  the  latter 
having  by  virtue  of  their  amoeboid  movements 
migrated,  during  the  space  of  time  the  tube  has 
been  left,  from  the  clot  into  the  serum.  With  the 
drop  of  serum  a  little  of  the  salt  solution  containing 
line  suspended  particles  of  the  pigment  is  thoroughly 
mixed,  and  the  preparation  is  then  covered,  the 
edges  of  the  cover-glass  being  surrounded  with  oil 
or  melted  paraffin,  to  prevent  evaporation.  When 
the  preparation  is  observed  it  will  be  seen  that  the 
white  corpuscles  have  quite  their  normal  appearance, 
whereas  the  colored  ones  for  the  most  part  exhibit 
folds  or  plaits  upon  their  surface,  having  undergone 
a  slight  shrinking,  owing  to  the  increased  density 
of  the  fluid  in  which  they  lie.  The  black  particles 
from  the  Indian  ink  are  scattered  irregularly  over 
the  fleld ;  and  although  they  may  here  and  there 
be  in  contact  with  the  white  corpuscles,  it  is  never- 
theless to  be  readily  made  out  that  the  substance  of 
the  latter  is  entirely  free  from  pigment.  But  after 
a  short  time,  if  the  corpuscles  are  closely  observed, 
it  will  be  seen  that  they  are  gradually  faking  into 


MIGRATION    OF    WHITE    CORPUSCLES.        45 

their  substance  the  black  particles  which  were  in 
contact  with  them  ;  and  when  by  their  amoeboid 
movements  from  one  place  to  another  they  are 
brought  into  contact  with  other  particles  these  also 
are  in  like  manner  absorbed.  On  careful  watching 
it  may  be  made  out  that  the  process  of  intussuscep- 
tion commences  by  the  throwing  out  of  processes 
which  surround  the  particle  to  be  taken  in,  and 
meet  and  coalesce  beyond  it ;  once  included  in  this 
manner,  the  grannie  afterwards  becomes  gradually 
carried,  presumably  by  the  movements  of  the  proto- 
plasm, more  towards  the  centre.  If,  after  observing 
the  preparation  in  this  way  for  half  an  hour,  it  be 
laid  aside  for  two  or  three  hours,  it  will  be  found  at 
the  expiration  of  that  time  that  many  of  the  white 
corpuscles  have  taken  in  a  large  number  of  the  black 
particles,  for  they  do  not  discharge  their  cargo,  but 
carry  it  about  in  their  movements  from  place  to 
place. 

This  property  of  the  white  blood-corpuscles  has  been 
used  in  pathological  investigations  to  determine  the 
source  of  the  corpuscles  of  pus,  which  resemble  the  white 
corpuscles  of  the  blood.  Insoluble  particles  were  in- 
jected into  the  bloodvessels  of  an  animal,  and  inflamma- 
tion was  induced  at  a  special  part:  it  was  found  that  the 
pus-corpuscles  which  accumulated  outside  the  blood- 
vessels in  the  inflamed  part  contained  similar  particles. 
The  corpuscles  must  consequently  have  come  from  the 
blood,  for  such  particles  have  not  the  power  of  passing 
through  the  coats  of  the  bloodvessels  unless  carried  by 
the  white  corpuscles. 

Preparation  10.  Migration  of  White  Cor- 
puscles.— The  process  of  migration  can  be  quite 
easily  seen  in  progress  in  transparent  parts  of 
animals,  and  will  be  afterwards  studied  when  the 
methods  of  observing  the  circulation  of  the  blood 
are  described.  But  for  exhibiting  the  active  migra- 
tion of  the  white  blood-corpuscles  nothing  is  more 
striking  than  the  examination  of  a  capillary  tube  in 


46 


PRACTICAL    HISTOLOGY. 


which  frog's  blood  has  been  collected  and  has  coagu- 
lated. A  high  power  is  to  be  employed,  and  the 
capillary  tube  must  therefore  be  very  line  indeed ; 
and  in  order  that  the  wall  of  the  tube  should  be  as 
thin  as  possible,  it  must  be  drawn  out  from  a  piece 
of  glass  tubing  half  an  inch  or  more  in  diameter. 
The  capillary  tube  is  filled  with  frog's  blood,  except 
near  the  ends ;  these  are  then  sealed  by  holding  them 
successively  in  the  flame  for  a  second  or  two ;  the 
tube  is  then  placed  in  a  drop  of  glycerine  on  a  slide, 
covered  with  a  thin  glass,  and  at  once  examined. 
The  object  of  the  glycerine  is  to  correct  in  some 
measure  by  its  high  refracting  power  the  effect 

Fig.  12. 


White  corpuscles  of  frog's  blood  migrated  from  clot.      Highly  mngnified. 
The  clot  has  shrunk  considerably  from  the  sides  of  the  capillary  tuba. 

upon  the  light  of  the  cylindrical  glass  tube.  After 
a  few  minutes  the  clot  is  seen  to  be  getting  smaller, 
and  a  layer  of  clear  serum  collects  between  it  and 
the  glass;  the  quantity  of  this  gradually  increases, 


ACTION    OF    REAGENTS.  47 

and  soon  one  or  two  white  corpuscles  begin  to  pro- 
ject from  the  surface  of  the  clot.  These  protrude 
more  and  more,  and  others  make  their  appearance, 
and  all  begin  to  throw  out  numerous  amreboid  pro- 
cesses, which  are  actively  advanced  and  retracted, 
and  by  aid  of  which  the  corpuscles  free  themselves 
entirely  from  the  shrinking  clot,  and  eventually 
become  free  in  the  surrounding  serum  (Fig.  12). 

Preparations  11,  12,  13,  14.  Action  of  Re- 
agents—Water, Acetic  Acid,  etc,— upon  the 
Frog's  blood-corpuscles. — The  action  of  reagents 
upon  the  white  blood-corpuscle  of  the  frog  is  ex- 
actly the  same  as  upon  the  mammalian  white  cor- 
puscle; upon  the  red  corpuscle  it  is  in  the  main 
similar,  but  in  some  cases  a  little  different,  the  diffe- 
rences partly  depending  upon  the  presence  of  the 
nucleus.  Thus  water  causes  both  the  substance  of 
the  corpuscle  and  the  nucleus  to  swell  up  and  become 
spheroidal,  and  extracts  the  colored  part  of  the 
corpuscles;  dilute  acetic  acid  brings  the  nucleus 
strongly  into  view,  and  decolorizes  the  rest  of  the 
corpuscle,  which,  however,  usually  retains  its  oval 
shape ;  tamric  acid  causes  the  colored  part  to  be 
exuded  from  the  stroma,  to  which  it  generally  re- 
mains attached  as  an  irregular  curdled  mass  (some- 
times the  colored  part  is  precipitated  around  the 
nucleus,  and  then  the  two  may  be  ejected  from  the 
stroma  together) ;  chloroform  vapor  causes  the  red 
corpuscles  to  become  decolorized,  and  arrests  the 
movements  of  the  white  corpuscles;  but  these,  if  not 
acted  on  for  too  long  a  time  or  by  too  strong  a  mix- 
ture of  chloroform  and  air,  resume  again  on  replac- 
ing the  vapor  by  pure  air.  All  these  reagents  are 
applied  in  precisely  the  same  manner  as  with  the 
mammalian  blood,  to  the  description  of  which  the 
reader  is  referred. 

The  observations  may  be  made  with  as  great  or 
with  greater  advantage  upon  the  blood-corpuscles  of 
the  newt,  which  are  larger  than  those  of  the  frog. 
The  blood  should  be  obtained  directly  from  the 


48  PRACTICAL  HISTOLOGY. 

heart,  and  not  by  merely  snipping  a  piece  off  the  tail 
and  collecting;  the  drop  which  exudes,  for  in  this  case 
it  is  very  apt  to  become  mixed  with  the  acrid  secretion 
from  the  cutaneous  glands.  There  are,  in  addition, 
two  reactions  for  which  newt's  blood  is  particularly 
well  adapted,  viz.,  the  actions  respectively  of  boracic 
acid  and  carbonic  acid  upon  the  red  corpuscles. 

Preparation  15.  Action  of  Boracic  Acid.— 
The  boracic  acid  is  used  in  solution  in  water  (two 
parts  per  cent.),  and  the  preparation  of  blood  having 
been  made  in  the  usual  way,  with  or  without  addi- 
tion of  salt  solution,  a  drop  of  the  boracic  acid  solu- 
tion is  placed  at  the  edge  of  the  cover-glass  and 
allowed  slowly  to  mingle  with  the  blood.  If  the 
first  stages  of  the  reaction  are  fortunately  observed 
it  will  be  seen  that  the  colored  part  of  the  corpuscle 
is  becoming  collected  towards  the  centre  of  the  cor- 
puscle and  accumulated  around  the  nucleus,  often 
remaining,  however,  at  first  adherent  here  and  there 
to  the  circumference  of  the  corpuscle,  and  shrinking 
away  at  the  intermediate  points,  so  as  to  present 
somewhat  of  a  stellate  figure.1  But  soon  it  is  en- 
tirely withdrawn  and  collected  around  the  nucleus, 
which  has  become  rounded,  and  is  nearly  concealed 
by  the  coloring  matter.  The  corpuscle,  now  decolor- 
ized, has  also  in  many  cases  become  circular,  and  the 
colored  nucleus  is  generally  shifted  to  one  side,  and 
eventually  altogether  extruded. 

Preparation  16, — To  investigate  the  action  of 
carbonic  acid  gas  the  blood  must  be  prepared  in  a 
moist  chamber,  like  that  used  for  chloroform  vapor 
(Fig.  11).  If  the  preparation  is  very  quickly  made 
the  nucleus  in  many  of  the  red  corpuscles  cannot  at 
first  be  distinguished,  for  in  the  entirely  unaltered 
state  it  possesses  as  nearly  as  possible  the  same  index 
of  refraction  as  the  rest  of  the  corpuscle.  But  when 
carbonic  acid  gas,  generated  in  a  suitable  apparatus 

1  Water  and  various  other  reagents  may,  in  the  first  instance, 
have  a  somewhat  similar  effect. 


WARMTH    ON    WHITE    CORPUSCLES.  49 

(Fig.  13,  6,  b'\  is  allowed  to  pass  into  the  moist 
chamber,  a  precipitate  of  one  of  the  constituents  of 
the  stroma  (probably  paraglobulin)  occurs  around 
the  nucleus,  and  the  outline  of  this  is  brought  into 
view,  whereas  if  the  carbonic  acid  is  speedily  replaced 
by  air,  which  may  be  effected  by  disconnecting  the 
tube  t  from  the  wash-bottle  6'  and  drawing  air 
through  it  by  the  mouth,  the  precipitate  is  redis- 
solved,  and  the  nucleus  is  again  made  to  disappear. 

Fig.  13. 


Apparatus  for  passing  carbonic  acid  gas  over  a  preparation  under  the 
microscope. 

ft,  bottle  containing  marble  and  hydrochloric  acid  ;  b',  wash-bottle:   t,  India- 
rubber  tube  conducting  the  gas  to  the  stage,  s. 

It  is  of  advantage  in  performing  this  experiment 
to  add  a  trace  of  moisture  to  the  blood  before  the 
observation;  this  may  be  most  conveniently  done 
by  breathing  two  or  three  times  on  the  preparation 
before  placing  it  over  the  ring  of  putty. 

Preparation  17.  Influence  of  warmth  on 
white  corpuscles  of  Amphibia. — The  action  of 
gentle  warmth  in  accelerating  the  movements  of  the 
5 


50 


PRACTICAL    HISTOLOGY. 


pale  corpuscles  of  the  frog  or  newt  may  be  investi- 
gated with  the  same  apparatus  as  was  used  for  the 
observation  of  mammalian  blood  at  the  temperature 
of  the  body.  But  it  will  be  found  that  if  the  tempe- 
rature be  allowed  to  rise  so  high  as  38°  0.  the  move- 
ments of  the  corpuscles  of  these  cold-blooded  animals 
will  soon  permanently  cease,  the  corpuscles  being 
killed  ;  so  that  unless  this  result  is  desired,  some  fat 
of  lower  melting-point  must  be  employed  to  indicate 
the  temperature  limit  which  is  not  to  be  exceeded. 

Preparation  18.  Action  of  Electric  Shocks. 
—For  this  a  slide  must  be  specially  prepared  by 
cementing  on  to  its  upper  surface  with  sbellac  var- 


Glass  slide,  with  two  strips  of  tin-foil,  one  of  which  pusses  round  to  the 
under  .surface,  where  it  rests  on  the  brass  stage  of  the  microscope  ; 
the  other  strip  is  isolated  from  the  stage,  and  may  be  connected  to 
the  outer  coating  of  a  Leyden  jar,  the  charge  of  wh'ch  is  ma.de  to 
pass  between  the  points  by  connecting  the  knob  of  the  jar  with  the 
brass-work  of  the  microscope.  Opposite  a,  a  small  piece  of  the  foil 
is  fixed  to  the  under  surface  of  the  slide,  so  that  this  end  shall  be 
level  with  the  other. 


ACTIOX    OF    ELECTRIC    SHOCKS    ON    BLOOD.       51 

nish  two  slips  of  gold-leaf  or  tin-foil,  with  pointed 
ends  which  almost  meet  in  the  middle  of  the  slide 
(Fig.  14,  A).  A  drop  of  blood  is  put  here,  the  cover- 
glass  is  placed  over  it,  and  the  portion  of  blood  which 
lies  between  the  points  is  brought  under  observation. 
Or  a  moist  chamber  may  be  employed,  the  cover- 
glass  used  having  previously  had  two  strips  of  tin- 
foil cemented  to^it  (Fig.  14,  B).  The  drop  of  blood, 
being  spread  out  in  a  thin  layer  between  their 
points,  is  quickly  inverted  over  the  ring  of  putty  and 
brought  under  observation.  The  tin-foil  slips  are 
kept  isolated  from  the  brass-work  of  the  microscope, 

Fig.  15. 


Apparatus  for  passing  electric  shocks  through  a  drop  of  blood,  which  is 
to  be  examined  in  a  moist  chamber.  The  tin-foil  slips  are  cemented 
near  their  points  to  the  under  surface  of  the  cover-glass,  and  their 
free  ends  are  clamped  to  isolated  metal  supports,  connected  by  wires 
to  an  induction  coil.  The  tin-foil  slips  are  isolated  from  the  brass 
stage  of  the  microscope  by  the  glass  slide  on  which  they  rest. 

and  are  so  arranged  that  the  charge  of  a  small 
Leyden  jar,  or  an  induced  current  of  electricity,  can 
be  passed  through  them  at  any  moment  (Fig.  15). 


52  PRACTICAL    HISTOLOGY. 

One  or  more  amoeboid  white  corpuscles  which  happen 
to  be  in  the  path  which  the  spark  must  take  in 
traversing  the  interval  between  the  points  are  kept 
in  view,  and  the  spark  is  then  allowed  to  pass.  The 
white  corpuscles  immediately  cease  moving,  with- 
draw their  processes,  and  become  rounded  in  shape; 
in  fact,  undergo  general  contraction.  But  if  only 
one  slight  shock  be  given  they  soon  recover  and 
resume  their  movements,  although  these  are  often 
somewhat  altered  in  character.  The  red  corpuscles 
are  but  slightly  if  at  all  affected  ;  but  if  a  succession 
of  shocks  be  transmitted  from  an  electric  machine 
or  an  induction-coil,  electrolytic  action  is  set  up  in 
the  fluid,  bubbles  of  gas  are  developed,  the  effects 
respectively  of  acids  and  alkalies  are  set  up  in  the 
neighborhood  of  the  tin-foil  points,  and  the  red 
corpuscles  undergo  changes  brought  about  by  these. 

Preparation  19.  Presence  of  Glycogen.— 
Many  of  the  white  corpuscles  contain  a  certain 
amount  of  glycogen,  or  animal  starch,  either  in  dis- 
tinct granules  or  in  a  more  diffused  form.  This 
substance  becomes  stained  of  a  reddish  mahogany 
color  by  solution  of  iodine,  and  may  thus  be  readily 
detected,  both  here  and  elsewhere.  The  solution  to 
be  used  is  made  by  dissolving  1  gramme  of  iodine 
dn  100  cc.  of  water,  which  contains  2  grammes  of 
iodide  of  potassium  in  solution. 

The  preparation,  preferably  of  frog's  or  newt's 
blood,  is  made  in  the  usual  way,  and  the  iodine 
solution  added  at  the  side  of  the  cover-glass.  The 
red  corpuscles  are  stained  of  an  intense  yellow,  but 
are  otherwise  little  altered,  except  that  the  nucleus, 
which  remains  unstained,  becomes  globular,  and 
bulges  out  at  either  surface  of  the  corpuscle.  The 
white  corpuscles  are  instantly  arrested  in  their 
movements  and  killed,  preserving  exactly  the  form 
which  they  exhibited  when  reached  by  the  iodine 
solution.  Being  of  less  specific  gravity  than  the 
latter,  they  tend  to  float  on  it ;  and,  if  the  layer  of 
fluid  is  thick,  must  be  sought  by  focussing  upwards 


HEMOGLOBIN    CRYSTALS.  53 

in  the  stratum  immediately  under  the  cover-glass. 
The  main  substance  of  the  corpuscle  is  uniformly 
stained  of  a  deep  yellow,  but  many  contain  groups 
of  mahogany-stained  granules,  and  from  others  are 
seen  to  exude  after  a  time  pellucid  drops  of  varying 
size,  which  become  tinted  of  a  mahogany  or  port- 
wine  color,  and  no  doubt  contain  glycogen. 

Preparation  20.  Crystals  obtainable  from  the 
Coloring  Matter  of  Blood. — The  hemoglobin  or 
coloring  matter  of  the  blood  may  be  obtained  in 
definite  crystals,  but  the  form  of  the  crystals  varies 
in  different  animals.  It  is  difficult  to  induce  the 
crystallization  in  human  blood ;  and  to  obtain  the 
crystals  readily  it  is  best  to  employ  the  blood  of  the 
rat. 

A  drop  of  this  is  mixed  with  an  equal  amount  of 
distilled  water,  covered,  and  observed  under  the 
microscope.  The  water  has  the  effect  of  extracting 
the  hemoglobin  from  the  corpuscles;  until  this  is 
done  no  crystallization  takes  place.  As  the  excess 
of  water  begins  to  evaporate  at  the  edges  of  the  cover- 
glass  small  needle-shaped  crystals  of  hemoglobin 
begin  to  appear,  either  singly  or  in  bunches,  which 
become  larger  and  larger  until  they  may  attain  a 
very  considerable  size. 

Preparation  21. — Crystals  may  also  be  readily 
obtained  from  guinea-pig's  blood,  either  by  the  same 
method  or  more  readily  perhaps  in  the  following 
way: — 

The  animal  is  decapitated,  and  the  blood  as  it 
flows  from  the  divided  vessels  is  vigorously  stirred 
with  a  small  bundle  of  wire,  to  remove  the  fibrin. 
A  small  quantity  of  the  whipped  blood  is  then 
mixed  with  about  one-third  its  volume  of  water, 
and,  a  drop  of  chloroform  being  added,  the  mixture 
is  thoroughly  shaken  up  for  a  minute  or  two.  This 
has  the  effect  of  discharging  the  hemaglobin  from 
the  corpuscles  into  the  surrounding  fluid.  A  small 
drop  is  now  placed  upon  a  slide  and  left  exposed  to 

5* 


54  PRACTICAL    HISTOLOGY. 

the  air  for  a  few  minutes.  It  becomes  thickened  by 
evaporation  and  dried  at  the  edges,  and  crystals, 
tetrahedral  in  form,  may  be  detected  in  it  with  a 
low  power  of  the  microscope.  The  preparation  may 
then  be  covered  and  examined  with  a  higher  power. 
The  crystals  increase  in  size  for  a  time,  and  new 
ones  continue  to  be  formed. 

Haemoglobin  crystals  will  not  keep  unaltered  for 
any  length  of  time. 

Preparation  22. — The  name  "  hsemin"  has  been 
given  to  certain  very  characteristic  crystals  which 
are  formed  at  the  expense  of  the  coloring  matter  of 
the  blood,  and  the  production  of  which  serves  as  an 
exceedingly  trustworthy  test  of  the  presence  of 
blood,  although  yielding  no  indication  of  the  kind 
of  animal  from  which  the  blood  has  been  obtained. 
To  see  them  a  very  small  quantity  of  blood  obtained 
from  the  finger  or  elsewhere  is  smeared  opon  a  glass 
slide  and  allowed  to  dry.  A  cover-glass  is  then 
placed  over  it,  and  a  drop  of  glacial  acetic  acid  is 
applied  from  a  pipette  to  the  edge  of  the  cover-glass 
and  allowed  to  run  under  by  capillary  attraction. 
The  slide  is  then  held  by  one  end  in  the  fingers,  and 
the  middle  is  gently  warmed  over  a  small  flame. 
As  soon  as  bubbles  begin  to  appear  in  the  fluid  the 
warmth  is  discontinued,  and  the  preparation  is  ex- 
amined with  a  high  power.  If  no  crystals  appear 
as  the  slide  cools  a  little  more  acid  is  added,  to  re- 
place that  lost  by  evaporation,  and  the  slide  is 
warmed  as  before,  and  on  cooling  again  examined. 
It  will  be  found  that  almost  all  over  the  preparation 
reddish-brown  short  prismatic  crystals,  disposed 
singly  or  in  groups,  will  have  made  their  appear- 
ance ;  most  of  them  are  very  minute,  but  they  may 
be  obtained  of  considerable  size  by  re-warming  the 
preparation  with  glacial  acetic  acid  once  or  twice. 

The  presence  of  a  chloride  is  necessary  for  the  for- 
mation of  these  crystals;  in  the  case  of  recent  blood 
the  chloride  of  sodium  which  it  naturally  contains 


HJSM IN    CRYSTALS.  55 

is  sufficient  for  the  purpose;  but  if  it  were  an  old 
blood-stain  whicb  one  had  to  deal  with,  in  which 
the  chlorides  may  have  been  washed  away,  it  would 
be  previously  requisite  to  mix  a  minute  quantity  of 
common  salt  with  the  stain  to  be  tested  in  order  to 
supply  the  deficiency. 


56  PRACTICAL    HISTOLOGY. 


CHAPTER   II. 

THE  EPITHELIAL  TISSUES. 

THE  epithelial  tissues  are  studied  with  regard  both 
to  the  character  and  form  of  the  individual  elements, 
and  the  relations  these  bear  to  one  another  and  to 
the  membranes  they  cover.  The  latter  class  of  ob- 
servation can  only  be  properly  made  by  the  study 
of  sections  of  the  various  organs  and  parts  where 
epithelium  is  found,  and  will  therefore  be  left  until 
the  method  of  making  these  is  explained.  The 
modes  of  isolating  and  studying  the  individual  cells 
will,  however,  be  best  described  in  this  place. 

Preparation  1.  Scaly  Epithelium,  Superfi- 
cial Layers. — If  a  little  of  the  saliva  which  moistens 
the  inside  of  the  cheek  be  gently  scraped  off  with  a 
small  spatula  or  with  the  finger-nail,  a  number  of  the 
superficial  cells  of  the  thick  stratified  epithelium 
which  is  here  met  with  will  be  brought  away, with 
it.  The  material  thus  obtained  is  placed  upon  a 
slide  and  a  cover-glass  put  over.  On  examining  the 
preparation  numerous  flattened  scaly  epithelium 
cells  will  be  seen,  either  entirely  isolated  or  in  little 
patches,  the  cells  in  a  patch  being  connected  together, 
with  their  edges  overlapping.  The  cells  are  of  con- 
siderable size,  each  with  a  nucleus  near  its  middle, 
small  in  comparison  with  the  size  of  the  cell ;  and 
the  substance  of  the  cell,  although  clear,  yet  contains 
a  number  of  scattered  granules.  Moreover,  lines 
may  often  be  seen  running  in  various  directions  over 
the  surface;  these  are  for  the  most  part  due  to  folds 
or  creases  of  its  substance.  Some  of  the  cells  may 
be  seen  edgeways,  and  then,  being  flattened,  will 
appear  narrow  and  linear;  but  on  touching  the 


STEATIFIED    EPITHELIUM.  57 

cover- glass  with  a  bristle  their  true  form  will  be 
apparent  as  they  tarn  over. 

In  addition  to  such  cells  as  these  a  certain  number 
of  much  smaller  rounded  cells  may  generally  be  seen 
in  the  saliva,  which  somewhat  resemble  the  white 
corpuscles  of  the  blood,  and,  like  them,  frequently 
exhibit  amoeboid  movements.  They  are,  in  fact,  to 
be  regarded  as  white  blood-corpuscles  which  have 
either  migrated  from  the  bloodvessels  into  the  sali- 
vary ducts  and  have  been  carried  into  the  mouth 
with  the  saliva,  or  have  corne  from  the  mucous 
membrane  covering  the  back  of  the  tongue  and  the 
tonsils,  which  is  very  rich  in  lymph-corpuscles.  The 
saliva  being  a  watery  fluid  they  are  swollen  out  by 
it,  and  with  a  good  microscope  it  may  be  observed 
that  the  granules  in  the  interior  of  the  corpuscles 
exhibit  the  Brownian  molecular  movement,  a  phe- 
nomenon which,  it  will  be  remembered,  was  exhi- 
bited by  the  white  corpuscles  of  the  blood,  as  a  first 
result  of  the  imbibition  of  water. 

Preparation  2.  Deeper  Layers. — There  are 
several  layers  of  the  above-described  large  flattened 
epithelial  cells  in  the  epithelium  of  the  mouth. 
Below  them  are  other  cells  smaller  and  of  a  more 
spheroidal  or  polyhedral  shape,  and  many  of  them 
having  ridges  and  spines  with  intervening  furrows 
upon  "their  surface.  To  obtain  these  isolated  it  is 
necessary  to  macerate  a  piece  of  any  membrane 
which  is  covered  by  a  similar  scaly  stratified  epithe- 
lium in  some  fluid  which,  while  it  softens  and  dis- 
solves the  intermediate  substance  which  cements 
the  cells  together,  may  preserve  their  natural  form, 
and,  at  the  same  time,  prevent  putrefaction  from 
appearing  in  the  tissue  which  is  undergoing  macer- 
ation. The  best  liquid  for  this  purpose  is  a  weak 
solution  of  bichromate  of  potash,  1  part  to  800  of 
water  (£  per  cent,).  The  portion  of  tissue  must  be 
small,  and  the  quantity  of  liquid  used  comparatively 
large.  A  piece  of  the  mucous  membrane  of  the 
mouth,  pharynx,  or  gullet  of  any  mammal  may  be 


58  PRACTICAL    HISTOLOGY. 

used ;  it  will  require  at  least  two  or  three  weeks' 
maceration  (for  in  this  kind  of  epithelium  the  cells 
are  very  closely  united),  and  the  bichromate  solution 
should  be  changed  every  third  day.  At  the  expira- 
tion of  the  time  stated  a  small  portion  of  the  epi- 
thelium is  scraped  oft'  with  the  point  of  a  knife  and 
placed  in  a  drop  of  water  upon  a  slide.  It  is  then 
broken  up  as  finely  as  possible  with  a  pair  of  mount- 
ed needles,  a  piece  of  a  hair  is  cut  off  and  placed  in 
the  drop,  and  the  cover-glass  is  superposed.  By  far 
the  majority  of  the  cells  seen  are  the  superficial  ones 
already  described  ;  but  others  may  be  found  which 
are  less  flattened  and  smaller  in  diameter,  and  have 
many  of  them  an  irregular  toothed  margin,  their 
surface  also,  as  may  be  seen  by  altering  the  fine 
adjustment  of  the  microscope,  having  on  it  linear 
or  punctated  markings.  These  are  the  cells  with 
ridges  and  spines  above .  mentioned  ;  they  will  be 
again  studied  in  sections  of  the  skin,  as  well  as  in 
sections  of  these  and  other  mucous  membranes  which 
are  covered  with  similar  epithelium. 

Preparation  3.  Horny  Layer  of  Epidermis. 
— If  a  very  small  shred  of  the  superficial  part  of  the 
epidermis  is  taken  from  any  part,  the  palm  of  the 
hand,  for  instance,  and  examined  in  water  under 
the  microscope,  no  indications  of  cellular  structure 
are  visible — nothing  but  a  hard  horny  confused  mass 
is  to  be  seen.  Remove  the  cover-glass  and  place  the 
shred  of  epidermis  in  a  drop  of  liquor  potassse.  It 
will  soon  swell  up  and  become  soft.  When  this  is 
the  case  return  it  to  the  water,  and,  after  breaking 
it  up  as  finely  as  possible  with  needles,  cover  and 
examine.  Numerous  spheroidal  cells  are  now  seen 
loose  in  the  preparation,  with  a  distinct  contour,  as 
if  inclosed  by  a  membrane,  but  without  a  nucleus. 
They  are,  in  fact,  the  scaly  cells,  which  have  become 
swollen  out  by  imbibition  of  water,  and  at  the  same 
time,  in  consequence  of  this  swelling  of  their  sur- 
faces, correspondingly  diminished  in  width. 

The  same  result  may  be  obtained  with  the  cells 


COLUMNAR    EPITHELIUM.  59 

which  form  the  nails;  and  also  with  the  flattened 
cells  from  the  mucous  membrane  of  the  mouth. 

Preparations  4,  5,  6,-- -The  Columnar  Epithe- 
lium is  most  characteristically  seen,  and  is  best 
studied  as  met  with  in  the  intestinal  canal.  It  must 
be  taken  from  an  animal  (rabbit)  quite  recently 
killed,  as  it  rapidly  undergoes  destructive  changes 
if  left  after  death  in  contact  with  the  intestinal  con- 
tents. When  the  intestine  is  cut  across  at  any 
part  in  a  recently  killed  animal  the  cut  edges  curl 
outwards,  and  a  little  of  the  mucous  membrane  is 
thus  exposed.  Two  very  small  portions  may  be 
snipped  off  this  and  placed,  one  in  a  few  drops  of  a 
one  per  cent,  solution  of  osmic  acid,  and  a  second 
in  iodized  serum.1 

These  two  portions  may  be  put  aside  for  the 
present  in  their  respective  fluids,  whilst  a  prepara- 
tion of  the  epithelium  in  the  fresh  condition  is 
made  and  examined.  For  this  purpose  slit  open  a 
piece  of  the  intestine,  wash  away  the  mucus  and 
intestinal  contents  by  allowing  a-little  of  the  serum 
from  the  animal's  blood  to  drop  upon  the  inner 
surface,  and  then,  with  the  end  of  a  clean  scalpel, 
gently  scrape  the  washed  surface  and  transfer 
what  is  brought  away  on  the  scalpel  to  a  drop  of 
fresh  serum  upon  a  clean  glass  slide,  and  cover  the 
preparation,  averting  the  pressure  of  the  cover- 
glass  by  means  of  a  piece  of  hair.  On  examining 
with  a  high  power  the  specimen  so  obtained,  nume- 
rous columnar  epithelium  cells  will  be  seen,  some 

1  This  is  prepared  from  the  amniotic  fluid  of  the  cow,  by  add- 
iug  to  it  a  crystal  or  two  of  iodine,  and  allowing  it  to  stand  for 
a  few  days,  with  frequent  agitation,  and  then  filtering  oft'  the 
clear  fluid  from  any  precipitate  that  may  have  formed.  Almost 
any  other  fresh  serous  fluid  can  be  used  instead,  but  the  amniotic 
fluid  is  generally  the  most  readily  obtained  in  sufficient  quantity, 
and  at  the  same  time  perfectly  clear  and  pellucid.  The  serous 
fluid  serves  to  macerate  tissues  which  are  immersed  in  it,  whilst 
preserving  as  nearly  as  possible  the  original  form  of  their  elements. 
The  purpose  of  the  iodine  is  to  prevent  putrefaction  ;  it  serves 
also  at  the  same  time  to  render  the  tissue-elements  slightly  firmer. 


60  PRACTICAL    HISTOLOGY. 

separate,  others  in  groups.  (It  will  be  found  advan- 
tageous in  examining  the  object  to  moderate  the 
illumination  by  the  employment  of  one  of  the  smaller 
holes  of  the  diaphragm  :  in  this  way  the  somewhat 
indistinct  outlines  of  the  tissue-elements  are  rendered 
plainer,  and  any  details  of  structure  can  generally 
be  more  readily  made  out ;  this  is  the  case,  indeed, 
with  most  preparations.)  In  the  separated  cells,  the 
conical  form  may  be  seen,  the  cell  either  tapering  at 
one  end  to  a  fine  point,  which  may  be  bifid,  or 
terminating  in  a  rounded  extremity.  The  general 
substance  of  the  cell  has  a  faintly  granular  appear- 
ance in  the  fresh  condition,  without  a  distinct  out- 
line, except  at  the  larger  end,  which  is  bounded  by 
a  strongly  refracting  thickened  margin,  in  which  a 
few  faint  striae  passing  from  without  inwards  may 
with  a  high  magnifying  power  be  made  out.  Near 
the  centre  of  the  cell  is  the  clear  oval  nucleus, 
bounded  by  a  distinct  line,  and  containing  generally 
one  nucleolus.  Globules  of  fat  of  varying  size  may, 
if  the  animal  were-  killed  during  digestion,  be  seen 
within  the  cell ;  they  are  recognized  by  their  strong 
refractive  power. 

Of  the  groups  of  cells  some  may  occur  in  which 
the  cells  are  seen  from  above  ;  the  collective  bases 
will  then  present  an  appearance  of  polygonal  areas 
intersected  by  lines  of  intercellular  substance;  in 
other  groups,  where  the  cells  are  seen  laterally,  their 
arrangement  with  regard  to  one  another  will  be 
observed. 

Some  cells  will  probably  be  seen  which  have 
acquired  a  peculiar  chalice-like  shape,  owing  to  the 
part  of  the  cell  near  the  free  surface  having  become 
swollen  out,  often  to  the  extent  of  bursting  away 
and  destroying  the  thickened  border.  These  are 
the  so-called  u  goblet  cells." 

The  other  two  portions  of  tissue  will  not  be  ready 
for  examination  for  some  days.  That  in  the  iodized 
serum  must  be  put  aside  in  a  cold  place,  in  a  well- 
stoppered  bottle.  At  the  expiration  of  a  week  a  pre- 


MODE    OF    FIXING    COVER-GLASS.  61 

paratiori  may  be  made  from  it  by  scraping  the  epi- 
thelial surface  as  before  and  mounting  the  product 
in  iodized  serum.  The  preparation  so  obtained  is 
similar  to  that  already  seen  of  the  fresh  epithelium, 
but  the  separated  cells  are  much  more  numerous, 
and  at  the  same  time  more  perfect,  for,  the  inter- 
cellular substance  which  unites  them  being  softened 
and  dissolved  by  maceration  in  the  serum,  the  cells 
are  more  readily  obtained  free. 

The  snip  which  was  placed  in  osmic  acid  must  be 
left  for  two  days ;  it  is  then  placed  in  water  for  an 
hour  or  two  to  remove  the  excess  of  the  acid.  To 
examine  the  tissue  remove  a  minute  portion  and 
break  it  up  as  finely  as  possible  with  needles  in  a 
drop  of  glycerine  on  a  slide.  The  cells  will  be  found 
to  exhibit  the  same  general  characters  as  regards 
form  and  appearance,  but  they  are  stained  of  a  dark 
gray  color,  and  in  consequence  appear  for  the  most, 
part  much  more  distinct.  Any  fatty  particles  which 
they  may  contain  are  colored  intensely  black.  This 
preparation  possesses  the  advantage  over  the  others 
that  it  can  be  preserved.  All  that  is  further  neces- 
sary is  to  apply  a  little  fixing  cement  of  some  sort 
around  the  edges  of  the  cover-glass. 

The  kind  of  fixing  cement  used  for  preparations 
mounted  in  glycerine  is  immaterial;  a  solution  of  hard 
Canada  halsam  in  chloroform,  dammar  varnish,  gold-size, 
or  Brunswick  black  may  be  employed  with  almost  equal 
advantage;  but  it  is  important  to  see  more  particularly 
to  two  points  before  applying  it.  The  first  is  that  there 
shall  be  just  enough  glycerine  in  the  preparation  to  fill 
up  the  space  between  the  cover-glass  and  slide,  neither 
too  much  nor  loo  little;  any  excess  must  be  removed 
with  blotting-paper.  At  the  same  time  one  or  two  small 
air-bubbles  in  the  glycerine  do  no  harm,  unless  they  are 
so  situated  as  to  obscure  the  tissue;  on  the  contrary, 
by  yielding  and  becoming  somewhat  compressed  when 
the  glycerine  tends  to  expand  from  being  exposed  (in 
summer,  for  instance)  to  a  warmer  temperature,  they 
may  tend  to  prevent  that  fluid  from  bursting  its  bonds 
6 


62  PRACTICAL    HISTOLOGY. 

and  exuding  through  the  inclosing  cement.  The  second 
point  to  be  attended  to  is  that  the  slide  where  the  cement 
is  to  be  applied  is  not  wetted  with  glycerine,  for  this 
would  prevent  the  adherence  of  the  cement. 

A  word  may  here  be  said  as  to  the  course  to  be  adopted 
should  any  glycerine  have  accidentally  got  on  to  the 
upper  surface  of  the  cover-glass.  No  attempt  should  be 
made  to  wipe  it  off'  at  once,  for  the  cover-glass  will  be 
pressed  upon  and  moved,  and  the  preparation  will  pro- 
bably be  crushed  and  spoiled  ;  but  time  should  be  allowed 
to  the  fixing  cement  to  become  thoroughly  hard,  and 
then  the  glycerine  can  be  removed  without  danger  to  the 
tissue  by  means  of  a  camel-hair  pencil  moistened  with 
distilled  water. 

For  the  present  it  will  be  sufficient  to  conclude 
the  study  of  epithelium  with  the  description  of  the 
best  modes  of  viewing  ciliated  epithelium,  and  of 
studying  the  action  of  various  reagents  upon  the 
ciliary  motion.  The  other  more  specialized  forms 
of  epithelium,  which  are  found  in  glandular  organs 
and  elsewhere,  will  he  seen  and  studied  when  the 
several  organs  and  parts  of  which  they  occur  are 
prepared. 

Preparation  7.  Ciliated  Epithelium  in  its 
living  state  may  he  readily  obtained  from  the  mouth 
and  gullet  of  the  recently  killed  Irog.  A  drop  of 
the  aqueous  humor  should  first  be  collected  by 
passing  a  capillary  glass  tube  through  the  cornea 
into  the  anterior  chamber  of  the  eye;  the  drop  is 
placed  upon  a  slide,  and  then,  the  frog's  mouth 
being  held  open  by  an  assistant,  the  roof  is  gently 
scraped  with  the  point  of  a  clean  scalpel,  so  as  to 
remove  the  adherent  mucus.  A  little  of  the  epithe- 
lium will  be  brought  away  with  this,  and  on  placing 
it  in  the  fluid,  and  covering  the  preparation  (taking 
the  precaution  of  previously  placing  a  hair  in  the 
drop),  the  cells  may  besought  for  with  a  high  power. 
For  the  most  part  they  will  be  collected  into  little 
groups  of  three  or  more,  the  cilia  being  in  active 
movement  and  producing  currents  in  the  liquid,  so 


CILIA    OF    MUSSEL.  63 

that  free  particles,  blood-corpuscles,  and  other  small 
objects  are  moved  along  in  it.  But  if  the  group  is 
small,  or  especially  if  entirely  isolated  cells  are  seen, 
it  will  generally  be  found  that  the  cilia  act  upon  the 
pieces  to  which  they  are  attached  like  little  paddles, 
moving  them  about  in  the  fluid.  The  cells,  it  may 
be  observed,  are  either  shortly  columnar  or  are 
spheroidal ;  the  nucleus  is  seldom  distinct,  because 
concealed  by  the  granular  nature  of  the  cell-proto- 
plasm. The  cilia  themselves  can  best  be  seen  when 
they  are  moving  languidly  or  when  their  motion  has 
altogether  stopped;  they  are  very  fine,  and  spring,  a 
number  together,  from  the  base  or  free  surface  of  the 
cell. 

Preparation  8.  Cilia  of  Mussel.— But  by  far 
the  most  convenient  object  for  the  study  of  ciliary 
motion  is  to  be  found  in  the  gill  of  the  common  sea- 
water  mussel  (Mi/stilus  editus).  Here  the  cilia  are 
very  large,  and  their  motion  will  go  on  unimpaired 
for  many  hours.  Hence  they  are  particularly  well 
suited  for  the  observation  of  the  action  of  most  of 
the  reagents  which  affect  ciliary  movement. 

One  or  more  mussels  may  readily  be  procured  at 
any  fishmonger's;  those  only  should  be  chosen  which 
remain  tightly  closed,  for  those  with  open  valves  are 
in  most  cases  already  dead.  One  of  the  shells  may 
then  be  forcibly  separated  by  means  of  a  knife,  when 
the  gills  (Fig.  16,  br)  will  come  into  view,  as  flattened 
expansions  of  a  yellowish  color,  covering  a  consider- 
able part  of  the  shell,  inside  its  lining  membrane m£ 
By  observing  carefully  it  may  be  noticed  that  they 
have  a  striated  aspect,  the  markings  passing  trans- 
versely to  their  length,  and  by  taking  up  a  small 
portion  with  a  forceps  it  will  further  be  seen  that 
this  striation  is  due  to  the  fact  that  the  gill  is  made 
up  of  a  number  of  little  bars  which  are  distinct  from 
one  another  for  the  greater  part  of  their  length. 
Take  now  a  small  piece  of  the  gill,  including  three 
or  four  of  the  bars,  and  placing  it  upon  a  side  in  a 
drop  of  the  sea- water  which  the  shell  always  contains, 


64  PRACTICAL    HISTOLOGY. 

separate  the  bars  one  from  another  by  means  of 
needles  ;  the  preparation  may  then  be  covered  and 
observed. 


Valve  of  mnesel.  showing  br,  br,  the  expanded  gills  or  branchiae,  which 
owing  to  the  little  bars  of  which  they  are  composed,  present  a  striated 
aspect. 

ml,  mantle  ;  m,  cut  adductor  moscle  ;  i,  mass  of  viscera  ;  the  dark  pro- 
jection just  above  is  the  foot. 

Each  of  the  bars  in  question  will  be  seen  to  be 
fringed  with  large  cilia,  which  are  set  at  an  appre- 
ciable distance  apart  along  nearly  its  whole  length, 
but  at  the  free  extremity  of  the  bar  are  much  more 
densely  arranged.  Those  in  this  situation  resemble 
in  appearance  the  cilia  of  the  frog's  mouth,  with  the 
exception  that  they  are  very  much  longer ;  and  like 
them  they  appear  to  spring  a  large  number  from 
each  cell,  whereas  the  others  are  stiff-looking  and 
obviously  thicker,  and  are  connected  at  their  base 
each  to  a  single,  comparatively  small  epithelium 
cell.  In  spite  of  these  differences  of  appearance  and 
attachment  the  two  kinds  seem  to  be  essentially 
alike  in  nature,  for  the  mode  in  which  they  move 
is  similar,  and  they  are  similarly  affected  by  re- 
agents. 

It  will  be  found  that  after  a  preparation  such  as 
that  just  described  has  been  made  for  several  hours 


ACTION    OF    REAGENTS    OX    CILIA.  65 

the  movement  will  have  become  somewhat  languid, 
and  then  the  manner  in  which  the  individual  cilia 
move  can  be  more  clearly  made  out.  The  prepara- 
tion can  be  used  also  for  the  study  of  those  agents 
which  tend  to  revive  and  stimulate  ciliary  motion, 
and  it  will  be  seen  that  it  is  precisely  the  ones  which 
most  accelerate  the  amoeboid  movements  of  the  white 
blood-corpuscles  that  have  the  most  marked  effect 
upon  the  cilia  also. 

Preparation  9.  Action  of  Warmth  upon 
Ciliary  Motion. — The  same  mode  of  applying  heat 
to  a  preparation  of  cilia  is  to  be  used  as  was  employed 
for  observing  the  effect  of  warmth  upon  the  blood 
(p.  30).  It  is  well,  after  inclosing  the  preparation 
with  oil  (or,  what  is  better,  with  melted  paraffin)  in 
the  manner  there  detailed,  to  put  it  aside  for  some 
time,  when  it  will  probably  he  found,  as  just  stated, 
that  the  movement  of  the  cilia  is  languid  or  alto- 
gether arrested.  On  now  gradually  warming  the 
preparation  the  motion  becomes  revived,  and  as  the 
heat  is  raised  becomes,  pan  passn,  faster  and  faster, 
until  a  point  is  reached  at  which  the  tissue  is  injured 
by  the  high  temperature,  when  the  movement  stops 
altogether,  and  is  not  again  resumed.  But  if  the 
experiment  be  stopped  short  of  this  point  and  the 
source  of  heat  removed,  it  will  be  seen  that,  con- 
versely, as  the  temperature  of  the  stage  falls,  the  rate 
of  movement  also  diminishes,  until,  when  again 
quite  cold,  the  cilia  may  again  almost  stop,  although 
they  can  be  made  to  resume  their  active  motion  on 
again  applying  warmth. 

"Preparation  10.  Action  of  Weak  Alkalies. 
— A  very  weak  solution  of  caustic  potash  in  salt 
solution,  like  that  which  was  used  in  investigating 
the  effect  of  weak  alkalies  upon  the  blood,  is  to  be 
applied  to  a  preparation  of  cilia  which  have  become 
somewhat  languid,  in  exactly  the  same  manner  as 
in  the  case  of  the  blood — by  allowing  a  little  of  the 
fluid  to  pass  in  at  the  edge  of  the  cover-glass  and 
diffuse  itself  with  the  "sea-water,  so  as  to  come 

6* 


66  PRACTICAL    HISTOLOGY. 

gradually  in  contact  with  the  slowly-moving  cilia. 
The  action  is  immediate ;  the  cilia  revive  and  vigor- 
ously lash  the  liquid  into  which  they  project,  but 
the  effort  is  soon  exhausted,  for  the  alkaline  liquid 
penetrating  the  cells  destroys  their  vitality,  and  the 
motion  of  their  cilia  stops  beyond  recovery.  The 
stimulant  action  is  not,  however,  peculiar  to  weak 
alkalies,  for  it  is  exhibited  also  by  acids  and  by 
many  other  substances  which,  applied  concentrated, 
would  instantly  destroy  the  tissue,  but  when  much 
diluted  tend  to  revive  and  for  a  time  maintain  accele- 
rated the  ciliary  motion. 

Preparation  11.  Action  of  Carbonic  Acid 
Gas. — This  reagent  is  to  be  applied  to  a  cilia  pre- 
paration in  the  gas-chamber  in  the  manner  directed 
for  the  investigation  of  its  action  upon  the  newt's 
blood-corpuscles  (p.  48).  Everything  being  ready, 
choose  a  part  of  the  preparation  where  the  cilia  are 
not  acting  very  vigorously,  and  whilst  still  watching 
allow  the  gas  to  pass  over  the  preparation.  Its  im- 
mediate action  is  seen  to  be  that  of  a  weak  acid — 
that  is  to  say,  the  rate  of  movement,  if  not  already 
at  its  fastest,  becomes  accelerated — but  as  soon  as 
the  oxygen  of  the  air  in  the  chamber  is  entirely  dis- 
placed by  the  continued  stream  of  carbonic  acid  the 
motion  ceases  altogether.  As  soon  as  this  result  is 
obtained  cut  off  the  stream  of  C02  and  reverse  the 
experiment  by  blowing  air  in  at  the  side-tube,  and 
thus  displacing  the  carbonic  acid  from  the  chamber. 
The  motion  will  almost  instantly  recommence.  This 
shows  that  it  was  the  absence  of  oxygen  and  not  the 
presence  of  C02  which  produced  the  stoppage  of 
movement ;  for  there  is,  of  course,  an  appreciable 
quantity  of  carbonic  acid  in  the  air  which  is  thus 
blown  from  the  mouth  into  the  chamber. 

Preparation  12.  Chloroform.— The  gas-cham- 
ber is  again  used  for  this  reagent,  the  apparatus 
being  arranged  in  the  way  previously  recommended 
(p.  39).  Choosing  a  part  of  the  preparation  where 
the  ciliary  motion  is  vigorous,  gently  blow  a  stream 


CILIATED    EPITHELIUM.  67 

of  the  mixture  of  air  and  chloroform  vapor  from  the 
bottle  into  the  moist  chamber.  The  cilia  become 
gradually  slower  and  eventually  stop.  Now  slip 
the  India-rubber  tube  off  the  bottle,  and  gently 
blow  air  through  the  chamber,  to  displace  the 
chloroform  vapor.  The  cilia  will  slowly  revive  on 
the  readmission  of  air,  and  will  soon  be  found  to 
work  as  vigorously  as  ever.  Like  that  with  the 
carbonic  acid  this  experiment  can  be  repeated  a 
number  of  times  with  a  like  result,  if  the  chloroform 
vapor  is  not  allowed  to  remain  too  long  in  contact 
with  the  preparation. 

-Preparation  13. — To  study  the  characters  of  the 
individual  cells  a  portion  of  membrane  which  is 
covered  by  ciliated  epithelium  is  macerated  in  some 
fluid  which  softens  and  dissolves  the  intercellular 
substance,  whilst  preserving  the  cells  themselves. 
The  best  for  this  purpose  is  a  weak  solution  of  bi- 
chromate of  potash  (1  in  800).  A  large  quantity  of 
this  must  be  employed,  and  the  tissue — a  piece  of 
the  trachea  of  a  rabbit  or  other  animal,  for  instance — 
is  left  in  it  for  about  forty-eight  hours.  With  the 
point  of  a  scalpel  a  little  of  the  epithelium  is  then 
gently  scraped  from  the  inner  surface,  and  being 
placed  in  a  drop  of  distilled  water  on  a  slide,  is 
broken  up  with  needles  as  finely  as  possible.  A 
small  piece  of  hair  is  placed  in  the  drop,  to  prevent 
the  soft,  delicate  cells  from  being  crushed  by  the 
weight  of  the  cover-glass.  This  is  now  superadded, 
and  the  preparation  carefully  examined  with  a  high 
power. 

Numerous  completely  isolated  cells  are  seen  float- 
ing in  the  liquid,  and  these  preserve  for  the  most 
part  their  natural  form  and  retain  their  cilia,  al- 
though the  latter  are,  of  course,  no  longer  in  motion. 
The  bright  border  through  which  the  cilia  appear 
to  pass,  the  faintly  granular  cell-substance,  the  clear 
nucleus  with  bright  nucleolus  in  the  middle,  and  the 
tapering,  branched  extremity  of  the  cell  are,  in  most, 
clearly  visible.  Besides  these  single  cell*,  others  are 


68  PRACTICAL    HISTOLOGY. 

present  which  are  still  united  in  groups  or  patches, 
in  which,  when  viewed  from  the  surface,  the  bases 
of  the  cells  have  a  mosaic  appearance.  Moreover,  a 
few  "goblet"  cells  may  here  and  there  be  met  with, 
no  doubt  produced  in  a  similar  manner  to  those 
found  amongst  the  columnar  cells  of  the  intestine 
and  elsewhere,  viz.,  by  a  swelling  up  of  the  mucus 
contained  within  the  cells  when  these  come  in  con- 
tact with  a  watery  fluid.  In  these  cases  the  base  of 
the  cell  is  ruptured  and  the  cilia  are  destroyed. 

Besides  the  ciliated  cells  certain  others  may  be 
seen  in  the  specimen  which  are  of  an  irregular  and 
usually  elongated  shape,  with  pointed  and  often 
branched  extremities,  and  are  destitute  of  cilia. 
They  lie,  in  the  natural  state,  between  the  ciliated 
cells,  but  their  position  can  only  be  properly  studied 
in  sections  of  the  mucous  membrane. 

If  it  be  wished  to  permanently  preserve  such  a 
preparation  as  that  now  under  description,  it  is 
necessary  first  to  stain  the  cells  somewhat  and  then 
to  substitute  glycerine  for  the  staining  fluid.  Either 
dilute  logwood  or  one  per  cent,  osmic  acid  solution 
may  be  used  for  staining  the  cells.  The  former 
colors  their  nuclei  very  intensely,  the  latter  gives  a 
uniform  gray  tint  to  the  cells.  The  coloring  fluid 
is  applied  in  the  following  manner :  A  little  is  taken 
up  into  a  pipette  and  a  drop  is  then  brought  in  con- 
tact with  one  edge  of  the  cover-glass.  The  pipette 
is  then  removed,  and  at  the  other  edge  a  small  piece 
of  filter-paper,  torn  into  a  triangular  shape,  is  placed, 
with  the  apex  of  the  triangle  touching  the  cover- 
glass,  so  as  to  draw  the  fluid  slowly  through.  When 
the  staining  fluid  has  replaced  the  water  in  which 
the  preparation  was  made,  the  filter-paper  is  removed, 
and  the  preparation  is  left  until  the  cells  appear 
sufficiently  colored,  a  little  more  fluid  being  occa- 
sionally added  if  there  seems  any  danger  of  the 
specimen  becoming  dry.  With  a  solution  of  log- 
wood, even  though  very  dilute,  a  few  minutes 


CILIATED    EPITHELIUM.  69 

suffices ;  the  osmic  acid  solution  should  be  allowed 
to  remain  an  hour  in  contact  with  the  cells.  The 
staining  fluid  is  replaced  by  water  in  the  same  way, 
and  finally,  the  filter-paper  being  removed,  a  small 
drop  of  glycerine  is  applied  at  one  border  of  the 
cover-glass  and  gradually  takes  the  place  of  the 
water  as  this  evaporates  at  the  edges.  In  a  day  or 
two  the  cover-glass  may  be  fixed  with  chloroform 
balsam. 


70  PRACTICAL    HISTOLOGY 


CHAPTER    III. 

CONNECTIVE  TISSUE. 

AREOLAR  TISSUE. — In  the  areolar  tissue  and  in 
connective  tissue  generally  there  are  several  parts 
which  present  themselves  for  study ;  and  in  order 
to  observe  each  to  the  greatest  advantage  different 
modes  of  preparation  are,  for  the  most  part,  requi- 
site. 

Preparations  1,  2.  The  Fibres  of  Areolar 
Tissue. — For  the  observation  of  the  fibrous  elements 
simply,  without  special  regard  to  their  arrangement 
or  relation  to  the  other  elements,  all  that  is  necessary 
is  to  place  a  small  portion  of  areolar  tissue,  taken 
from  any  part,  on  the  centre  of  a  glass  slide,  just 
moistened  with  salt  solution,  and  with  clean,  sharp 
needles,  separate  it  as  finely  as  possible  into  filamen- 
tous shreds.  Then,  before  there  is  time  for  the  pre- 
paration to  become  dry,  place  a  drop  of  salt  solution 
on  a  cover-glass  and  invert  this  over  the  tissue.  The 
object  of  using  but"  very  little  fluid  to  prepare  the 
tissue  in  is  to  prevent  the  filaments  from  running 
together  and  becoming  entangled  when  released  from 
the  needles. 

In  a  preparation  so  made,  nothing  is,  as  a  rule, 
apparent  save  the  wavy  bundles  of  connective  tissue 
fibrils,  these  when  much  developed  obscuring,  by 
their  effect  upon  the  light,  the  elastic  fibres  and  cor- 
puscles of  the  tissue.  But  if  now  a  second  prepara- 
tion be  made  in  precisely  the  same  way,  except  that, 
in  place  of  salt  solution  simply,  salt  solution  con- 
taining one  part  of  acetic  acid  in  200  is  placed  upon 
the  cover-glass,  and  if  then  the  object  is  immediately 
examined  with  a  high  power,  it  is  seen  that  the 


CONNECTIVE    TISSUE    CORPUSCLES.  71 

fibrils  which  compose  the  bundles  have  become  in- 
distinct, whilst  the  bundles  are  much  swollen,  except, 
it  may  be,  at  intervals  here  and  there.  At  the  same 
time  certain  other  fibres,  almost  equally  fine  but 
more  sharply  defined  than  the  white  fibrils,  and 
always  running  singly,  never  in  bundles,  come  into 
view.  These  are  the  elastic  fibres.  If  one  of  them 
be  followed  for  a  short  distance,  it  will  probably  be 
seen  that  it  sooner  or  later  gives  off  a  branch  which 
unites  it  with  a  neighboring  fibre,whereas  the  white 
fibrils  never  show  any  disposition  to  branch  or  unite 
with  one  another,  but  those  in  each  bundle  maintain 
from  end  to  end  a  perfectly  parallel  course.  The 
elastic  nature  of  the  filaments  which  are  brought 
into  view  by  acetic  acid  is  shown,  in  such  a  prepara- 
tion as  we  are  describing,  by  the  fact  that  wherever 
in  the  process  of  teasing  the  tissue  they  have  become 
broken  across,  the  fibres  have,  by  the  recoil  from  the 
stretching  to  which  they  were  submitted  before  the 
rupture  occurred,  been  thrown  into  bold  curves,  es- 
pecially marked  near  the  broken  extremities,  which 
are  often  recurved.  That  this  curved  or  coiled  ap- 
pearance of  the  elastic  fibres,  although  highly  char- 
acteristic, and  always  observable  when  the  tissue  is 
thus  prepared,  is,  however,  not  a  natural  one,  is 
shown  by  the  fact  that,  as  will  immediately  be  de- 
scribed, when  precautions  are  taken  to  preserve  as 
much  as  possible  the  normal  arrangement  of  the 
tissue  elements,  the  elastic  fibres  are  seen  to  pursue 
a  rectilinear  course. 

Preparation  3.  The  Corpuscles  of  the  Areolar 
Tissue. — To  demonstrate  the  cells  or  connective 
tissue  corpuscles  the  preparation  is  made  more 
methodically.  A  film  as  thin  as  possible  must  be 
obtained  for  observation,  so  as  to  avoid  the  necessity 
of  tearing  the  tissue.  Such  films  are  naturally  pre- 
sent in  the  areolar  tissue  of  most  parts,  and  may  be 
seen  when  the  organs  which  it  connects  are  gently 
drawn  asunder  from  one  another,  as,  for  instance, 
when  the  skin  is  raised  and  reflected  from  the  sub- 


72  PRACTICAL    HISTOLOGY. 

jacent  fascise  and  muscles.  The  most  convenient 
source  of  such  a  delicate  film  is  to  be  found  in  the 
exquisitely  thin  and  transparent  tissue  which  invests 
and  lies  between  the  muscles  of  the  fore-limb  of  the 
rabbit  and  guinea-pig.  The  tissue  in  this  situation, 
especially  if  taken  from  a  young  animal,  is  devoid, 
of  fat  and  not  so  completely  overridden  by  the  bun- 
dles of  white  fibrils  but  that  the  elastic  fibres  and 
the  connective  tissue  corpuscles  can  be  made  out 
even  without  the  addition  of  reagents.  The  mode 
of  preparation  is  as  follows  :— 

The  animal  having  been  killed  by  bleeding,  the 
skin  is  snipped  through  around  the  upper  part  of  the 
fore-limb  and  is  then  forcibly  reflected  from  the  limb. 
In  this  operation  care  must  be  taken  to  avoid  be- 
sprinkling the  subjacent  parts  with  the  cut  hairs  of 
the  animal.  A  piece  of  the  tissue  over  or  between 
the  muscles  is  then  seized  with  the  forceps  and 
snipped  off  with  sharp,  fine  scissors..  The  snipped-ofi 
tissue  shrinks  immediately  around  the  end  of  the 
forceps  and  appears  very  unsuited  for  microscopical 
examination.  But  place  it  on  a  clean  slide,  without 
the  addition  of  any  fluid,  and  with  a  pair  of  mounted 
needles  endeavor,  by  drawing  out  first  this  corner 
and  then  that,  to  again  reduce  the  gelatinous-looking 
piece  to  the  condition  of  a  thin  film,  and  it  will  be 
found  that  this  can  be  effected  without  much  diffi- 
culty, for  when  not  floated  up  by  fluid  the  thin  edges 
of  the  film  tend  to  stick  to  the  glass,  and  cease  to 
shrink  away  from  the  position  to  which  they  are 
drawn  by  the  needles.  At  the  same  time,  whilst  it 
is  important  not  to  add  fluid  to  that  which  naturally 
moistens  the  piece  of  tissue,  it  is  equally  important 
never,  during  the  whole  process  of  stretching,  to  let 
the  film  become  actually  desiccated,  for  this  would 
altogether  ruin  the  tissue  for  microscopic  purposes. 
The  best  way  to  prevent  such  an  untoward  result 
from  happening  is  to  breathe  now  and  then  on  the 
object  whilst  it  is  being  prepared;  by  so  doing  need- 
less haste  will  be  avoided  and  more  time  and  pains 


CONNECTIVE    TISSUE    CORPUSCLES.  73 

can  be  taken  for  the  complete  display  of  the  film. 
This  being  effected,  a  cover-glass  (which  should  have 
been  previously  cleaned  and  placed  in  readiness)  is 
taken,  wetted  on  its  under  surface  with  a  drop  of 
salt  solution,  and  quickly  superposed  over  the  film 
of  tissue,  which  is  thus  prevented  from  shrinking  up 
again  into  a  shapeless  mass.  The  fibres,  both  white 
(in  wavy  bundles  of  various  sizes)  and  the  elastic, 
and  corpuscles  may  now  be  carefully  observed,  at 
first  with  the  usual  high  power  and  afterwards  with 
the  highest  obtainable,  and  some  of  the  corpuscles 
should  be  sketched.  Moreover,  search  may  be  made 
for  lymph  or  pale  blood-corpuscles,  a  very  few  of 
which  are  generally  to  be  found  in  the  connective 
tissue;  they  are  readily  distinguished  from  the  fixed 
corpuscles  of  the  tissue  by  their  small  size — small, 
obscure,  and  generally  multiple  nuclei — and  espe- 
cially their  amoeboid  movements,  of  which  it  is  pro- 
bable no  trace  will  be  apparent  even  to  the  most 
assiduous  observation  in  the  connective  tissue  cells 
proper. 

Preparation  4. — Although  both  corpuscles  and 
elastic  fibres  may  be  seen  in  a  preparation  of  this 
kind  made  with  an  indifferent  fluid,  they  are  better 
seen  if  the  white  fibres  are  acted  upon  by  acetic  acid, 
and  still  better  if  this  action  is  combined  with  that 
of  some  staining  reagent,  so  that  the  corpuscles  are 
brought  more  prominently  into  view.  Moreover, 
the  preparation  admits  of  being  permanently  pre- 
served in  glycerine  after  such  a  method  of  treat- 
ment. Up  to  a  certain  stage  the  procedure  is  the 
same  as  that  above  described,  but  instead  of  placing 
salt  solution  upon  the  cover-glass,  before  inverting 
it  over  the  film,  a  solution  of  acetic  acid  (one  per 
cent.)  colored  by  the  addition  to  it  of  one-third  its 
volume  of  logwood-alum  solution,  is  employed.  The 
object  is  examined  as  quickly  as  possible  after  the 
application. 

The  connective  tissue  corpuscles  can  probably  be 
made  out  already  in  the  thinner  parts  of  the  prepa- 


74  PRACTICAL    HISTOLOGY. 

ration,  with  their  clear  oval  nuclei  and  the  flattened 
irregular  area  occupied  by  their  cell  substance.  In  a 
few  minutes  the  nuclei  will  be  tinted  by  the  logwood, 
and  will  then  show  up  much  more  prominently  ; 
but  to  get  the  cell-bodies  sufficiently  colored  it  will 
be  necessary  to  leave  the  staining  solution  half  an 
hour  or  more  in  contact  with  the  preparation.  Mean- 
while, to  obviate  the  effects  of  evaporation,  a  con- 
siderable drop  of  the  coloring  fluid  should  be  placed 
on  either  side  in  contact  with  the  edges  of  the  cover- 
glass.  The  excess  of  fluid,  moreover,  has  a  tendency 
to  raise  the  latter  slightly  from  the  lilm  of  tissue, 
and  in  this  way  a  more  ready  access  of  fresh  coloring 
fluid  is  permitted.  When  it  is  found  on  examina- 
tion that  the  corpuscles  are  properly  stained,  the 
solution  may  be  drawn  off  by  a  slip  of  filter-paper 
applied  to  the  edge  of  the  cover-glass  on  one  side, 
whilst  to  the  other  a  drop  of  distilled  water  is  applied, 
the  logwood  being  for  the  most  part  in  this  manner 
rinsed  away.  The  filter-paper  is  then  removed,  and 
a  drop  of  glycerine  placed  in  contact  with  the  edge; 
this,  as  the  water  evaporates,  will  slowly  diffuse  it- 
self under  the  cover-glass  and  take  its  place.  It  is 
well  to  put  the  preparation  aside  until  the  following 
day,  when  the  process  will  be  completed  ;  all  that  is 
then  necessary  is  to  draw  a  camel-hair  pencil  wetted 
with  chloroform  balsam  around  the  edge  of  the 
covering-glass,  so  as  to  fix  it  securely  in  its  place. 

In  specimens  treated  with  acid  there  may  be  observed 
a  constricting  ring  at  intervals  along  the  course  of 
some  of  the  connective  tissue  bundles,  an  appearance 
which  has  long  been  familiar  to  histologists,  but  the 
cause  of  which  is  not  yet  clearly  determined,  some  sup- 
posing it,  from  its  resistance  to  the  action  of  acids,  to  be 
of  the  nature  of  elastic  tissue  ;  others  that  the  appearance 
is  caused  by  the  process  of  a  corpuscle  enwrapping  the 
bundle ;  the  latter  opinion  being  mainly  founded  on 
the  fact  that  the  ring  becomes  stained  by  certain  re- 
agents which  also  color  the  cells.  For  example,  as  shown 
by  Kanvier,  the  constricting  ring  is  tinted  red  by  picro- 


METHOD    OF    LOCALIZED    (EDEMA.  75 

carminateof  ammonia,  whilst  the  elastic  fibres  are  colored 
yellow. 

Preparation  5. — In  most  of  the  larger  animals 
(e.g.  the  dog),  the  connective  tissue  is,  in  the  adult, 
so  densely  pervaded  by  the  bundles  of  while  fibres 
as  to  render  it  impossible  to  obtain  a  film  delicate 
enough  for  easy  observation  without  tearing  the 
texture  with  needles,  and  thereby  distorting  the 
cellular  elements.  Or,  on  the  other  hand,  it  may  be 
desired,  even  in  those  animals  in  which  such  delicate 
tissue  as  that  the  preparation  of  which  has  just  been 
described  is  found,  to  obtain  a  specimen  from  a  part 
where  the  connective  tissue  is  not  naturally  extended 
in  so  advantageous  a  manner  for  preparation  and 
observation.  In  these  cases  the  following  method 
may  be  employed  with  advantage : — 

In  a  recently  killed  dog  a  flap  of  skin  is  dissected 
back,  and  a  Pravaz  or 'other  hypodermic  syringe 
(Fig.  17),  provided  with  a  fine  canula,  having  been 

Fig.  17. 


Pravaz  syringe  for  interstitial  injections;  c,  the  cauula.  The  syringe  is 
glass  with  ebonite  fittings  ;  the  canula  is  a  fine  perforated  steel 
needle  set  in  ebonite.  Natural  size. 

previously  filled  with  salt  solution,  the  point  of  the 
canula  is  inserted  underneath  the  layer  of  connec- 
tive tissue  which  is  most  superficial  on  the  reflected 
portion  of  skin,  and  a  little  of  the  fluid  is  forced  out. 
This  does  not  immediately  diffuse  itself  uniformly 
through  the  loose  areolar  tissue,  but  remains  for  a 
short  while  at  the  same  place,  forming  a  little  bulla 
of  liquid  bounded  and  covered  in  by  a  film  of  tissue, 


76  PRACTICAL    HISTOLOGY. 

the  thickness  of  which  depends  upon  the  depth  to 
which  the  canuhi  was  inserted.  If  it  does  not 
appear  thin  enough  a  second  attempt  should  be  made 
at  another  spot.  Then,  before  the  bulk  has  time  to 
subside — that  is  to  say,  before  the  fluid  has  time  to 
diffuse  itself  through  the  meshes  of  the  tissue — snip 
off  the  whole  projection  with  a  single  cut  of  a  pair 
of  scissors,  which  for  this  purpose  should  be  particu- 
larly sharp  and  clean,  and  transfer  the  snipped- oft' 
portion  to  a  clean  slide.  Here  it  may  either  be  at 
once  covered  in  salt  solution  and  examined  without 
reagents,  or  may  be  treated  with  acetic  acid,  logwood, 
&c.,  as  in  the  mode  of  preparation  just  described. 

Preparation  6. — A  modification  of  the  above 
method  consists  in  injecting  into  the  tissue  a  solu- 
tion of  gelatine  instead  of  a  salt  solution.  The  gela- 
tine solution  is  made  by  taking  some  very  clear 
French  gelatine,  allowing  it  to  soak  for  an  hour  in 
water,  and  then,  after  pouring  off'  all  the  excess  of 
that  fluid,  placing  the  soaked  gelatine  in  a  beaker 
over  a  water  bath  until  it  is  entirely  melted  in  the 
water  which  it  had  imbibed.  The  syringe  is  then 
warmed  by  immersing  it  for  a  minute  or  two  in 
warm  water,  and  is  filled  with  the  gelatine  solution, 
and  a  little  of  this  is  injected  into  the  subcutaneous 
connective  tissue,  so  as  to  produce  a  bulla  like  that 
made  by  the  salt  solution.  In  cold  weather  the 
gelatine  will  set  almost  immediately ;  in  warm 
weather  the  process  may  be  accelerated  by  surround- 
ing the  bulla  with  small  lumps  of  ice. 

When  the  gelatine  is  quite  firm  sections  of  it  are 
to  be  made  with  a  razor.  As  they  are  cut  they  may 
be  placed  in  salt  solution. 

Before  mounting  them  it  is  well  to  stain  the 
specimens,  and  one  of  the  best  staining  fluids  for 
the  purpose  is  a  watery  solution  of  magenta.  This 
colors  the  elastic  fibres  strongly,  the  corpuscles  dis- 
tinctly, and  the  bundles  of  white  fibrils  slightly, 
while  the  gelatine  which  was  injected  and  of  course 
occupies  all  the  interstices  is  hardly  stained  at  all. 


METHOD    OF    GELATINE    (EDEMA.  77 

The  time  of  immersing  the  sections  varies  of  course 
with  the  strength  of  the  fluid,  but  this  should  not 
be  too  highly  colored,  and  it  can  be  then  seen  with- 
out much  difficulty  when  the  sections  are  sufficiently 
stained.  They  are  subsequently  placed  in  water  for 
a  minute  or  two  to  remove  the  excess  of  magenta 
prior  to  transferring  them  to  a  slide.  A  drop  of 
glycerine  is  now  added  and  the  cover-glass  laid  on, 
after  which  the  slide  is  gently  warmed  over  a  small 
flame  or  otherwise  until  the  gelatine  in  the  sections 
just  melts  so  as  to  allow  the  cover-glass,  which  was 
probably  tilted  up  somewhat  owing  to  the  thickness 
of  the  sections,  to  settle  down.  The  specimen  may 
then  be  examined,  and  if  satisfactory,  may  be  pre- 
served, the  preparation  being  completed  after  a  day 
or  two  by  fixing  the  edges  of  the  cover-glass  with 
chloroform  balsam. 

By  the  modes  of  preparing  connective  tissue  already 
described  the  bundles  of  white  fibres,  the  elastic  fibres, 
and  the  corpuscles  are  brought  under  observation,  and 
it  would  seem  at  first  sight  that  these  of  themselves 
entirely  constitute  the  tissue.  But  in  considering  the 
nature  of  the  films  obtained — that  they  are,  namely,  con- 
tinuous over  a  greater  or  less  area — it  is  clear  that  the 
presence  of  fibres  and  cells  is  not  alone  sufficient  to 
account  for  the  lamina?  which  are  spread  upon  the  slide. 
And,  indeed,  by  closely  observing  the  preparation  it  will 
be  apparent  that  there  is  pellucid  substance  uniting  every- 
thing together,  through  which  the  fibres  run,  and  in  which 
the  corpuscles  lie  embedded.  There  is,  it  is  true,  a  diffi- 
culty in  making  this  out  in  most  parts,  in  consequence 
of  its  extreme  clearness,  and  the  fact  that  its  refractive 
index  is  little  different  from  that  of  the  watery  fluid  the 
tissue  is  examined  in  ;  moreover,  in  the  logwood  pre- 
parations the  intermediate  substance  remains  entirely 
unstained.  Nevertheless,  towards  the  edge  of  the  pre- 
paration, where  a  comparison  can  the  better  be  made 
with  the  surrounding  fluid,  the  fact  that  such  a  clear 
intermediate  substance  does  really  exist  will  be  suffi- 
ciently evident ;  and  the  more  so  if  the  cover-glass  be 
slightl}'  moved  or  one  edge  be  gentlv  pressed  down  with 

7* 


78  PRACTICAL    HISTOLOGY. 

a  needle.  But  we  possess  in  the  silver  method  of  Reck- 
linghausen  a  ready  means  of  demonstrating  its  existence 
in  an  obvious  manner;  for  the  ground  substance  (or 
intercellular  substance)  of  the  connective  tissue,  and, 
indeed,  of  almost  every  other  tissue  in  the  body,  possesses 
the  distinctive  property  of  reducing  the  salts  of  silver 
under  the  action  of  light,  so  that  the  metal  is  deposited 
in  it  either  in  a  free  state  or,  more  probably,  in  combina- 
tion. The  effect  of  this  deposition  is,  that  the  ground 
substance  becomes  stained  of  a  color  varying  with  the 
intensity  of  the  light  employed,  and  with  oilier  conditions, 
from  a  light  brown  to  a  brownish-black.  The  iibrous 
elements  participate  for  the  most  part  in  this  staining, 
and  are  frequently,  especially  when  the  preparation  has 
been,  as  is  usual  with  silver  preparations,  mounted  in 
glycerine,  indistinguishable  from  the  ground  substance 
through  which  they  course,  and  which  also  unites  the 
white  fibres  into  the  bundles  which  they  form.  The 
cellular  elements,  on  the  contrary,  remain  absolutely 
unstained,  and,  moreover,  after  the  action  of  the  silveV 
salt  are  no  longer  affected  by  those  staining  fluids  which 
otherwise  have  a  particular  aSinity  for  them  ;  it  is  there- 
fore no  longer  possible  to  bring  them  into  view.  Where- 
ever,  then,  a  cell  is  situated,  there  appears  after  the 
reduction  of  the  silver  nothing  but  a  white  patch  upon 
or  in  the  brown  ground;  and  if,  as  is  not  unfrequently 
the  case,  several  flattened  cells  may  have  occurred 
together  with  their  edges  in  juxtaposition,  the  group 
appears  as  a  larger  white  patch  intersected  by  dark  lines, 
these  representing  a  small  amount  of  intercellular  sub- 
stance between  the  individual  cells.  The  appearance  is 
similar  to  what  is  observed  in  an  epithelial  tissue  after 
the  silver  treatment,  for  in  this  the  intercellular  substance 
is  always  very  small  in  amount.  Such  an  arrangement 
of  connective  tissue  cells  is  on  this  account  designated 
"  epithelioid."  The  white  patches,  then,  in  the  silvered 
preparation  of  connective  tissue  represent  either  depres- 
sions on  the  surface  of,  or  actual  cavities  within,  the 
matrix  or  ground  substance,  containing  cells,  which 
themselves  are  not  visible,  so  that  the  white  patches  are 
termed  the  cell-spaces,  or  (recalling  the  analogous  case 
of  bone)  the  lacuna  of  the  connective  tissue. 

It  is  the  more  appropriate  to  give  them  a  special  desig- 


NITRATE    OF    SILVER    PREPARATIONS.       79 

nation,  because,  as  may  be  made  out  by  a  careful  compari- 
son of  specimens  of  connective  tissue  from  the  same  part, 
some  prepared  with  logwood  or  chloride  of  gold,  to  show 
the  cells,  others  with  silver,  to  show  the  cell-spaces,  the 
cell-spaces  are  in  many  cases  distinctly  larger  than  the 
cells,  and  may  also  he  of  a  somewhat  different  figure; 
they  are  not  necessarily,  therefore,  as  has  sometimes  been 
supposed,  and  as  is  no  doubt  the  case  with  the  clear  parts 
of  a  silvered  epithelium,  merely  the  cells  left  white.  The 
difference  in  the  relative  size  of  the  cell-spaces  and  the 
contained  cells  obtains  no  doubt  more  frequently,  or  at 
least  can  be  more  readily  made  evident,  in  the  firmer 
varieties  of  connective  tissue,  where  the  ground  substance 
is  everywhere  pervaded  with  fibrous  bundles,  and  has  in 
consequence  lost  its  soft  and  yielding  nature,  which  other- 
wise permits  it  to  adapt  itself  more  readily  to  the  shape 
of  the  cells.  Moreover  it  must  be  remembered  that  the 
natural  course  of  the  lymph  in  the  tissue  is  around  the 
cells,  between  them  and  the  inclosing  ground  substance; 
and  when  from  any  cause  that  fluid  is  in  excess  the  effect 
will  be  that  the  cell-spaces  are  more  distended  and  appear 
preternatu rally  large. 

This  much  having  been  said  in  order  to  explain 
the  appearances  produced  by  the  silver  method  of 
treatment — appearances  which  have  been  called  in 
question,  and  their  value  as  yielding  evidence  of  any 
constant  structure  in  the  tissues  altogether  denied 
by  histologists  of  considerable  eminence,  but,  as  it 
must  now  be  admitted,  without  sufficient  grounds — 
the  best  mode  of  applying  it  to  ordinary  connective 
tissue,  such,  for  instance,  as  the  subcutaneous,  may 
now  be  described. 

Preparation  7. — The  skin  of  a  recently  killed 
rabbit  or  guinea-pig1  having  been  stripped  off  one  of 

1  These  ammals  are  selected  because  there  is  likely  to  be  less 
fat  in  the  subcutaneous  tissue  than  in  that  of  the  cat  or  dog  or 
other  animals  commonly  used  in  the  laboratory.  It  is  important 
to  remember  that  any  tissue  which  is  to  be  submitted  to  the  silver 
method  must  be  fresh  and  unacted  upon  previously  by  any  re- 
agent whatever,  since  this  would  entirely  prevent  the  desired 
effect;  moreover,  if  blood  have  accidentally  got  on  to  the  part,  it 
must  first  be  rinsed  away  by  distilled  water. 


80  PRACTICAL    HISTOLOGY. 

the  limbs,  this  is  disarticulated  at  the  proximal  joint, 
and  is  rinsed  for  a  second  or  two  in  a  beaker  of  dis- 
tilled water,  in  order  to  wash  away  any  blood  or 
lymph,  which  might  happen  to  be  on  the  surface, 
and  which  would  cause  a  granular  precipitate  with 
the  nitrate  of  silver  solution  The  latter,  a  solution 
of  1  part  of  the  salt  to  200  of  distilled  water,  is 
then  either  poured  over  the  surface  or  dropped  on  it 
from  a  pipette.  After  two  or  three  minutes  the 
silver  solution  is  quickly  washed  off  by  a  stream  of 
distilled  water,  and  the  limb  is  then  at  once  placed 
in  a  beaker  of  spirit,  and  exposed  to  direct  sunlight, 
or,  failing  this,  to  bright  diffused  daylight.  In  a 
few  minutes  in  the  sunlight,  and  after  a  longer  time 
in  diffused  daylight,  the  silvered  surface  will  have 
acquired  a  uniform  brownish  tinge  to  the  naked  eye. 
When  the  color  is  strongly  marked,  it  is  as  well  to 
remove  the  beaker  from  the  light,  lest  the  prepara- 
tion become  too  darkly  stained.  The  limb  should 
be  allowed  to  remain  in  the  spirit  twenty-four  hours; 
at  the  expiration  of  this  time  it  is  placed  in  a  dish, 
and,  by  the  aid  of  fine  forceps  and  scissors,  a  piece 
of  the  superficial  stained  layer  is  dissected  off  under 
spirit.  In  doing  this,  care  must  be  taken  not  to 
drag  at  all  upon  the  membrane  which  is  removed, 
so  as  to  throw  it  into  creases.  The  piece  is  then 
transferred  to  water,  and  floated  on  to  a  clean  glass 
slide  writh  the  browned  surface  of  the  membrane 
uppermost.  The  slide  is  then  carefully  removed 
from  the  water,  the  portion  of  tissue  being  kept  flat 
on  its  upper  surface ;  and  most  of  the  superfluous 
water  having  been  wiped  away,  a  cover-glass,  on 
which  a  drop  of  glycerine  has  been  placed,  is  in- 
verted over  the  preparation.  Before  putting  on  the 
cover-glass  it  is  well  to  examine  the  object  under 
a  low  power,  in  order  to  make  sure  of  the  absence 
of  folds  and  creases  or  specks  of  dust  upon  it :  if 
any  such  be  seen  they  must  be  carefully  removed 
with  a  needle.  Indeed,  it  may  be  recommended  as 
a  golden  rule  in  making  histological  preparations 


CELL -SPACES    OF    CONNECTIVE    TISSUE.      81 

never  to  put  the  cover-glass  in  its  place  until  a 
glance  at  the  ohject  under  a  low  power  of  the  micro- 
scope has  certified  the  absence  of  any  marked  imper- 
fection :  if  this  be  attended  to,  the  time  will  often 
be  saved  which  would  otherwise  be  spent  in  mount- 
ing worthless  specimens. 

These  silver-preparations  of  the  subcutaneous  tissue 
vary  considerably  in  appearance  according  to  the  part  of 
the  limb  from  which  they  are  taken,  and  it  is  in  man}7 
cases  difficult  full}7  to  comprehend  the  meaning  of  what 
is  seen,  so  that  it  will  be  better  perhaps  to  omit  for  the 
present  this  preparation,  especially  as  the  one  following 
furnishes  a  very  ready  and  clear  means  of  demonstrating 
the  cell  spaces  of  connective  tissue. 

Preparation  8. — The  connective  tissue  which 
covers  the  tendons  of  the  superficial  flexor  digitorum 
of  the  ox's  foot  as  they  run  through  sheaths  formed 
by  the  tendons  of  the  deep  flexor  is  much  more  easily 
prepared  by  the  silver  method  than  the  looser  kinds 
like  the  subcutaneous  just  described.  A  piece  of 
such  a  tendon  is  taken,  rinsed  in  distilled  water  to 
remove  the  synovial  fluid  which  covers  it,  and  treated 
with  silver  solution  in  the  way  just  described;  and 
is  then,  after  washing.,  placed  in  the  light  in  strong 
spirit.  It  soon  becomes  brown,  when  it  may  be  re- 
moved from  the  light;  and  after  remaining  twenty- 
four  hours  in  the  spirit  it  is  easy,  with  a  sharp  knife 
or  razor  wetted  with  spirit,  to  obtain  a  thin  surface 
section.  This,  after  being  immersed  for  a  minute 
or  two  in  water  to  get  rid  of  the  spirit,  is  mounted 
in  glycerine  with  the  browned  surface  uppermost. 
It  should  present,  if  successful,  an  extremely  charac- 
teristic and  beautiful  image  of  white  branched  cell- 
spaces,  single  or  in  groups,  upon  a  brown  ground. 

In  both  the  preparations  last  described  it  is  possi- 
ble to  show  the  nuclei  of  the  corpuscles  which  lie 
in  the  cell-spaces  by  subsequent  staining  with  log- 
wood. Sometimes  they  are  visible  even  without 
this  treatment. 


82  PRACTICAL    HISTOLOGY. 

Preparation  9.  Elastic  Network.— The  pro- 
portion of  elastic  fibres  varies  considerably  according 
to  the  part  from  which  the  connective  tissue  under 
examination  is  taken.  Some  serous  membranes  con- 
tain a  large  number  of  elastic  fibres;  and  since  they 
are  readily  spread  out  in  their  natural  condition  the 
network  which  these  fibres  form  by  their  branchings 
and  conjunctions  is  easily  made  evident.  One  of 
the  best  objects  for  this  purpose  is  to  be  found  in  the 
rabbit's  mesocolon.  A  piece  of  this,  moistened  with 
a  little  salt  solution,  may  be  spread  out  as  flat  as 
possible  upon  a  slide,  and  a  drop  of  dilute  acetic  acid 
(one  per  cent.)  having  been  placed  on  a  cover-glass, 
this  is  inverted  over  the  tissue.  The  white  fibrils 
quickly  swell  up  and  become  indistinct,  whilst  the 
elastic  network  comes  clearly  into  view.  The  pre- 
paration may  be  made  permanent  by  putting,  as  in 
former  preparations,  a  drop  of  glycerine  at  the  edge 
of  the  cover-glass,  and  after  this  has  had  time  to  dif- 
fuse itself,  cementing  the  edges  with  chloroform  bal- 
sam as  usual. 

Preparation  10.  Elastic  Tissue.— The  elastic 
ligaments  are  to  be  regarded  merely  as  connective 
tissue  structures  in  which  the  elastic  elements  of  the 
tissue  preponderate.  There  is  always  a  quantity  of 
ordinary  areolar  tissue  amongst  the  elastic  fibres 
even  here,  but  not  sufficient  to  obscure  them,  espe- 
cially as  they  are  generally  of  larger  size  as  well  as 
in  greater  number  than  elsewhere.  It  is  sufficient, 
in  order  to  see  them,  simply  to  teaze  out  a  portion  of 
the  ligamenta  subflava  of  the  vertebrae  or  other 
elastic  tissue  in  water  or  salt  solution.  If  it  be 
desired  to  keep  the  preparation  it  can  be  mounted 
in  glycerine. 

Preparation  11.  Section  of  Elastic  Fibres. — 
To  observe  the  shape  of  the  fibres  a  transverse  section 
may  be  made  of  a  piece  of  ligamentum  nuchae  of  the 
ox,  in  which  the  fibres  are  extremely  large. 

In  order  to  obtain  the  requisite  firmness  for  cut- 
ting, place  a  small  piece  of  the  ligament  in  a  quantity 


PREPARATION    OF    TENDOX.  83 

of  two  per  cent,  solution  of  bichromate  of  potash  for 
fourteen  days  ;  then  place  in  water  for  two  or  three 
hours,  and  transfer  to  spirit ;  in. a  day  or  two  sec- 
tions across  the  direction  of  the  fibres  may  be  made 
with  a  razor,  the  surface  of  which  should  be  wetted 
with  spirit.  The  sections  are  to  be  placed  in  water 
for  a  minute  or  so, and  finally  mounted  in  glycerine. 

Preparation  12.  Fibrous  or  Tendinous  Tis- 
sue.— This  may  be  examined  by  separating  a  small 
shred  from  a  tendon  or  ligament,  and  teazing  it  out 
as  finely  as  possible  into  its  constituent  bundles. 
The  operation  is  conducted  with  the  aid  of  needles, 
the  tissue  being  placed  in  a  drop  of  salt  solution,  and 
it  is  first  examined  in  this,  being  afterwards  treated 
with  dilute  acetic  acid  and  logwood  (see  Prep.  4). 
But  it  is  a  troublesome  matter  to  make  the  separa- 
tion fine  enough  without  disturbing  too  much  the 
arrangement  of  the  cellular  elements  of  the  tissue. 
Fortunately  we  can  obtain,  from  the  tail  of  the  mouse 
or  rat,  tendons  which  are,  FO  to  speak,  naturally  dis- 
sociated ;  for  excessively  fine  tendons  run  along  the 
whole  length  of  the  tail,  and  can  readily  be  drawn 
out,  needing  no  further  manipulation  than  is  neces- 
sary to  place  them  advantageously  under  the  micro- 
scope. The  following  is  the  mode  of  procedure  : — 

Preparation  13. — In  a  recently  killed  mouse  the 
tail  is  seized  about  half  an  inch  from  the  tip  between 
the  thumb  nail  and  fore-finger  of  the  right  hand  ; 
and  the  delicate  skin  being  partially  nipped  through 
and  the  vertebral  column  broken  at  this  point  by  the 
pressure  of  the  nail,  it  will  be  found  quite  easy,  the 
base  of  the  tail  being  fixed  by  the  left  fore-finger 
and  thumb,  to  separate  the  end  altogether  and  drag 
it  away  from  the  remainder  of  the  tail.  In  doing 
so  it  will  be  found  that  the  minute  tendons  which 
are  attached  near  the  tip,  owing  to  their  compara- 
tive toughness  and  strength,  are  not  broken  through 
at  the  spot  in  question,  but  are  dragged  out  of  the 
channels  in  which  they  run,  and  may  in  this  way 
be  obtained  in  the  form  of  a  bundle  of  exquisitely 


84  PRACTICAL    HISTOLOGY. 

fine  silky  threads,  which  are  to  be  immediately  im- 
mersed in  a  glass  dish  of  salt  solution.  Now  cut 
away  two  or  three  of  the  fine  threads  with  sharp, 
clean  scissors,  and  seizing  them  by  one  end  with  fine 
forceps,  or  leading  them  with  a  needle-point,  float 
them  on  to  a  glass  slide  which  is  held  immersed  in 
the  fluid,  and  is  then  carefully  lifted  out.  After 
arranging  the  minute  tendons  as  nearly  straight  as 
possible  on  the  slide,  and  blotting  up  must  of  the 
superfluous  salt  solution  or  allowing  it  to  run  oft', 
place  a  short  piece  of  hair  beside  them,  to  avert  the 
pressure  of  the  cover-glass,  which  is  now  placed  over 
the  middle  of  the  threads  in  such  a  way  that,  since 
they  are  considerably  longer  than  the  width  of  the 
cover-glass,  their  ends  project  beyond  on  either  side. 
The  object  of  this  is  to  permit  them  to  be  drawn 
straight  with  needles  should  the  superposition  of  the 
cover-glass  have  displaced  them.  These  ends,  more- 
over, since  they  are  exposed  to  the  air,  soon  dry  and 
stick  to  the  slide,  so  that  subsequent  treatment  with 
reagents  does  not  tend  to  displace  the  tendons,  which 
are  thus  maintained  in  an  extended  condition.  Ex- 
amined thus  in  salt  solution,  little  is  visible  beyond 
the  slightly  wavy,  closely  packed  white  iibrils,  col- 
lected, as  longitudinal  streaks  seen  here  and  there 
indicate,  into  a  few  indistinct  bundles.  But  allow 
a  little  dilute  acetic  acid  (1  part  of  the  glacial  acid 
to  200  of  salt  solution)  slowly  to  pass  under  the 
cover-glass,  and  a  remarkable  change  becomes  appa- 
rent. "As  the  acid  reaches  the  tendons,  they  slowly 
swell  up  and  become  more  transparent,  the  fibrils 
becoming  indistinct ;  and  now  chains  of  small  oblong 
faintly  granular  cells,  each  with  a  clear  nucleus 
situated  near  one  end  of  the  cell,  and  often  opposite 
that  of  a  neighboring  cell,  come  into  view.  These 
are  the  tendon-cells,  the  corpuscles  of  the  fibrous 
connective  tissue  ;  only  the  central  thicker  portion 
of  each,  which  lies  in  the  interstice  between  three 
or  more  tendon  bundles,  is  seen  at  present  ;  the  thin 
lamellar  prolongations,  which  extend  between  two 


CELLS    OF    TENDON.  85 

tendon  bundles,  are  too  delicate  to  be  made  out 
without  staining.  In  some  of  the  chains  a  bright 
longitudinal  line  is  to  be  seen  on  each  cell ;  this  ap- 
pearance is  merely  produced  by  a  lamellar  prolonga- 
tion of  this  sort,  which  happens  to  extend  vertically 
to  the  plane  under  observation. 

After  the  action  of  the  acid  has  been  prolonged 
for  some  time,  the  cells  gradually  lose  their  dis- 
tinctness, and  eventually  can  with  difficulty  be 
made  out,  although  the  iibres  are  more  swollen  and 
indistinct  than  ever.  But  if  a  little  of  the  colored 
acetic  acid  which  was  used  for  the  subcutaneous 
tissue  (Prep.  4)  is  allowed  to  run  under  the  cover- 
glass,  first  the  nuclei,  and  then  the  bodies  of  the 
cells  and  their  prolongations,  become  colored,  whilst 
the  fibres  remain  unstained,  just  as  in  the  parallel 
case  of  the  areolar  tissue. 

When  the  coloration  is  sufficiently  deep,  the 
staining  fluid  may  be  replaced  by  water,  and  this 
in  the  usual  way  by  glycerine  ;  and  finally,  the  edges 
of  the  cover-glass  being  cemented,  the  preparation 
can  be  permanently  preserved.  Examined  with  a 
high  power,  the  tendon  cells  now  appear  in  the  suc- 
cessive horizontal  planes  as  quadrangular  flattened 
bodies,  thickest  near  the  middle,  and  gradually 
shading  off  at  either  side,  and  marked  with  one  or 
more  dark  lines  running  longitudinally,  which  are, 
in  fact,  the  bright  lines  to  which  attention  was  pre- 
viously drawn,  and  which  have  now  become  stained. 
To  show  that  these  are  actual  flattened  extensions 
of  the  cell,  and  not  mere  markings,  it  is  necessary  to 
compare  the  appearances  presented  by  a  transverse 
section. 

Another  method  of  displaying  the  cells  of  tendon  is 
that  originally  employed  by  Ranvier.  One  or  two  of  the 
small  tail  tendons  are  placed  on  a  slide,  and  their  ends 
are  fixed  with  paraffin,  so  as  to  keep  them  extended.  A 
few  drops  of  one  per  cent,  solution  of  picrocanninate  of 
ammonia  are  then  placed  upon  them  and  left  for  half  an 
hour,  after  which  the  picrocarmme  is  washed  away  with 
8 


86  PRACTICAL    HISTOLOGY. 

distilled  water  mid  the  tendons  are  mounted  in  glycerine, 
acidulated  with  acetic  acid. 

The  picrocarmine  is  made  by  adding  to  a  saturated 
solution  of  picric  acid  a  strong  solution  of  carmine  in 
ammonia,  to  saturation,  evaporating  the  mixture  to  one- 
fifth  part  of  its  bulk,  allowing  to  cool,  filtering  from  the 
deposit  which  occurs,  and  evaporating  the  filtrate  to  dry- 
ness  over  a  water-bath  ;  when  the  picrocarmine  is  left  as 
a  crystalline  powder  of  an  ochre-red  color. 

Preparation  14.  Transverse  section  of  Ten- 
don.— To  obtain  this,  it  is  best  to  take  a  large 
tendon,  tor  it  is  much  easier  to  get  transverse  sec- 
tions of  sucli  a  one,  and  in  all  essential  points  of 
structure  it  is  quite  similar  to  the  minute  tendons 
which,  for  the  sake  of  convenience,  we  have  just 
been  employing.  A  piece,  then,  of  any  tendon, 
large  enough  to  be  grasped  by  the  fingers,  is  placed 
in  strong  spirit  for  a  day  or  two.  This  gives  it  a 
very  hard, 'horny  consistence,  and  it  is  easy,  with  u 
sharp  knife  or  razor  wetted  with  the  spirit,  to  get  one 
or  two  thin  sections  from  the  end.  These  are  placed 
on  a  slide,  a  drop  of  the  acidulated  logwood  is  added 
and  left  in  contact  with  them  until  they  are  suffi- 
ciently stained.  It  is  then  carefully  washed  away 
by  a  drop  or  two  of  water  applied  from  a  pipette ; 
and  finally  a  little  glycerine  is  placed  upon  a  cover- 
glass,  which  is  then  inverted  over  the  preparation. 

It  will  be  seen  that  the  tendon  is  divided  into 
fasciculi  by  septa  of  areola  tissue,  the  corpuscles  of 
which  (seen  edgeways)  are  brought  into  view,  being 
stained  by  the  logwood  ;  it  will  further  be  observed 
if  the  sectional  area  of  one  of  the  smaller  fasciculi 
is  attentively  examined  with  a  high  power,  that  it 
again  is  divided  (although  incompletely)  into  several 
still  smaller  bundles  by  the  branching  processes  of 
deeply  colored  stellate  bodies  situate  at  the  angle  of 
junction  between  three  or  more  such  bundles,  and 
extending  a  greater  or  less  distance  between  the 
neighboring  bandies.  These  stellate  bodies,  with 
their  processes,  are  the  tendon-cells  with  the  himcl- 


ADIPOSE    TISSUE.  87 

lar  extensions,  as  seen  in  section  ;  the  smaller  fasci- 
culi, which  are  separated  by  areolar  tissue,  corre- 
sponding with  the  whole  of  one  of  the  caudal  ten- 
dons of  the  mouse  or  rat. 

Preparation  15.  Cell-spaces  of  Tendon. — To 
show  the  cell-spaces  in  which  the  above-mentioned 
tendon-cells  are  contained: — 

As  before,  break  off  the  end  and  draw  out  some  of 
the  tendons  of  a  mouse's  tail  (that  previously  used 
will  still  yield  a  sufficient  number).  Then  hold  the 
tendons  in  a  shallow  dish  of  distilled  water,  and 
with  a  medium-sized  camel-hair  pencil  brush  them 
firmly  from  end  to  end.  six  or  eight  times.  Remove 
them  now  from  the  water,  and  immerse  them  in  a 
large  watch-glass  of  nitrate  of  silver  solution  (J  per 
cent.)  for  fifteen  minutes;  then  place  in  a  glass  ves- 
sel of  water  and  expose  to  sunlight.  As  soon  as  they 
are  well  browned,  pieces  may  be  cut  off,  laid  straight 
in  water  upon  a  slide,  covered,  and  the  water  then 
drawn  off  and  replaced  by  glycerine;  after  which 
the  cover-glass  may  be  fixed  in  the  usual  way. 

Preparation  16.  Epithelioid  covering  of  Ten- 
dons.— The  object  of  first  brushing  the  tendons  is 
to  remove  the  layer  of  flattened  cells  which  covers 
the  surface  of  each,  and  which,  if  allowed  to  remain, 
prevents  the  silver  solution  from  properly  acting 
upon  the  deeper  parts  of  the  tissue.  To  show  this 
layer,  another  set  of  tendons  may  be  treated  with 
the  silver  solution  in  a  similar  way,  but  with  a 
minute's  immersion  and  without  previously  brush- 
ing them,  when  it  will  probably  be  found  that  the 
superficial  epithelioid  stratum  is  alone  apparent. 

Preparation  17.  Adipose  Tissue.— The  sim- 
plest way  of  showing  the  fat  vesicles  is  by  teasing 
out  a  small  portion  of  the  tissue  in  a  drop  of  salt 
solution;  taking  the  precaution  of  putting  a  narrow 
slip  of  blotting-paper  on  either  side  to  avert  the 
pressure  of  the  cover-glass. 

But  a  far  better  way  of  demonstrating  the  struc- 
ture of  the  tissue  generally,  consists  in  the  employ- 


88  PRACTICAL    HISTOLOGY. 

ment  of  the  method  of  interstitial  injection  with 
gelatine  (see  Preparation  6,  p.  76).  The  gelatine  is 
injected  into  the  interior  of  one  of  the  fat-lumps, 
and  sections  are  made  and  treated  in  the  way  as 
described.  By  this  method  the  fat-cells  are  some- 
what separated  from  one  another,  and  all  their  parts, 
as  well  as  the  intermediate  tissue  and  bloodvessels, 
are  much  better  displayed. 

Preparation  18.  Membrane  of  the  fat-cell. 
— To  show  this  distinctly  Ranvier's  method  may  be 
recommended.  An  interstitial  injection  is  made 
with  a  weak  solution  of  nitrate  of  silver  (1  in  1000), 
with  a  minute  portion  of  the  fat-lump  thus  rendered 
cedematous  is  removed  with  scissors,  transferred  to 
a  slide  and  covered.  Many  of  the  fat  cells  exhibit 
the  envelope  and  nucleus  separated  by  a  distinct 
space  from  the  fat-drop.  The  silver  solution  would 
appear  to  have  penetrated  by  endosmosis  and  to  have 
become  collected  between  the  fat-drop  and  its  inclos- 
ing membrane. 

Preparation  19. — To  complete  the  study  of  adi- 
pose tissue,  the  fat-cells  should  be  observed  in  pro- 
cess of  development.  For  this  purpose  a  preparation 
of  the  subcutaneous  tissue  from  a  part  where  fat  is 
being  deposited  may  be  obtained  from  the  moderately 
advanced  foetus  of  any  mammal.  The  new-born  rat 
is  especially  to  be  recommended,  since  in  its  subcu- 
taneous tissue  there  are  generally  to  be  found,  not 
only  cells  which  are  in  every  stage  of  fat-deposition, 
but  others  in  addition  which  exhibit  the  formation 
of  bloodvessels  and  the  simultaneous  formation  with- 
in the  same  cells  of  red-blood  corpuscles.  The  mode 
of  preparation  is  very  simple,  all  that  is  necessary 
being  to  strip  the  skin  from  the  back,  snip  oft'  with 
scissors  a  little  of  the  gelatinous-looking  tissue  from 
the  borders  of  a  tract  where  the  fat  is  already  partly 
deposited,  and  to  place  the  portion  so  obtained  in  a 
drop  of  salt  solution  upon  a  slide,  and  cover  it  with 
a  thin  glass. 


CARTILAGE.  89 


CHAPTER    IV. 

CARTILAGE. 

Articular  Cartilage. — This  is  to  be  studied  in 
sections  made  both  parallel  and  vertical  to  the 
surface. 

Preparation  1. — From  an  animal  that  has  just 
been  killed  remove  one  of  the  limb-bones,  with  its 
articular  ends,  and  with  a  clean,  sharp  scalpel  or 
razor  take  a  slice,  as  thin  as  possible,  off'  the  carti- 
lage, and  quickly,  before  it  has  time  to  become  dry, 
transfer  the  piece  to  a  drop  of  serum  upon  a  slide, 
place  a  cover-glass  over  the  preparation  and  examine 
with  a  high  power.  Turning  the  attention  more 
particularly  to  the  cartilage-cells,  the  arrangement 
of  these  in  groups  in  the  faintly  granulous  matrix 
will  be  noticed.  Each  cell  is  seen  to  be  provided 
with  a  clear  round  nucleus,  which  in  some  speci- 
mens of  articular  cartilage  is  so  large  proportion- 
ately that  it  may  be  mistaken  by  an  inexperienced 
observer  for  the  whole  cell.  In  reality,  however, 
the  cell-substance  is  represented  by  the  clear  material 
(or  containing,  at  most,  a  few  highly  refracting 
granules)  which  lies  around  the  nucleus  and  entirely 
fills  the  cavity  or  space  in  the  cartilaginous  matrix 
in  which  the  cell  lies.  But  now  replace  the  serum 
by  distilled  water,  drawing  the  former  away  by 
means  of  a  piece  of  blotting-paper  placed  at  one  edge 
of  the  cover-glass,  and  allowing  a  drop  of  water  from 
a  pipette  to  run  under  at  the  opposite  edge  (see  Fig. 
18),  and  the  picture  soon  changes.  Examine  the 
cells  at  the  borders  of  the  slice,  for  these  are  first 
reached  by  the  water.  It  will  be  seen  that  the  clear 
cell-substance  begins  to  be  separated  from  the  matrix, 

b* 


90 


PRACTICAL    HISTOLOGY. 


and  acquires  a  jagged  outline,  fluid  collecting  in  the 
interspace  which  is  now  left.  This  clear  fluid  is 
stated  by  some  to  be  water  which  has  permeated 
the  matrix,  and  p-issing  into  the  cell-space  has 

Fig.  18. 


pushed,  as  it  were,  the  cell  substance  away.  But 
the  more  probable  explanation  is  that  the  cartilage- 
cells,  by  virtue  of  a  certain  amount  of  vital  contrac- 
tility which  they  retain,  shrink  on  the  application 
of  the  water,  as  they  do  on  the  application  of  many 
other  reagents  and  on  the  passage  of  an  electric 
shock,  and* that  the  clear  fluid  which  collects  around 
them  is  expressed  from  their  protoplasm  as  it  con- 
tracts. "Whatever  the  explanation  may  be,  the  effect 
is  this:  that  the  now  jagged,  shrunken  cell-body 
assumes,  instead  of  the  clear  aspect  which  in  the 


ARTICULAR    CARTILAGE.  91 

fresh  condition  it  presented,  a  coarsely  granular 
appearance,  so  much  so  indeed  that,  the  nucleus 
which  was  previously  so  apparent  is  now  entirely 
obscured.  Moreover,  as  already  indicated,  the  car- 
tilage-cell no  longer  fills  the  cell-space  in  which  it 
lies.  The  cells  always  undergo  this  change  after 
death,  unless  the  tissue  has  been  treated  with  some 
reagent  which  prevents  its  occurrence. 

In  preparing  a  specimen  of  cartilage  with  the 
object  of  permanently  preserving  it,  our  aim  should 
be,  as,  indeed,  with  every  tissue,  to  obtain  it  in  a 
condition  and  form  as  nearly  as  possible  approaching 
that  which  it  had  whilst  living.  There  are  nume- 
rous reagents  which,  in  place  of  acting  like  water 
and  causing  contraction  of  the  cartilage-cells,  fix 
them  in  the  form  they  present  during  life.  Amongst 
these  osmic  acid  may  be  mentioned  first,  as  the  most 
generally  valuable  reagent  which  we  possess  for  this 
purpose,  since  it  acts  in  like  manner  upon  nearly  all 
the  tissues.  But  it  will  not  here  be  recommended 
for  the  purpose  of  preserving  the  tissue  of  cartilage 
in  consequence  of  its  costliness,  and  from  the  fact 
that  other  and  cheaper  reagents  serve  the  purpose 
equally  well.  One  special  value  it  certainly  has, 
however  ;  namely,  in  showing  that  the  little  granules 
in  the  protoplasm  of  the  cartilage-cell  are  many  of 
them  of  a  fatty  nature,  for  they  are  blackened  by 
the  reagent.  A  one  per  cent,  solution  of  alum,  and 
a  saturated  solution  of  picric  acid  (both  recom- 
meded  by  Ranvier),  preserve  the  cells  of  cartilage 
admirably.  But  one  of  the  best  and  most  conve- 
nient reagents  for  the  purpose  is  a  weak  solution  of 
chromic  acid  (1  part  to  600  of  water). 

Preparations  2  and  3. — The  articular  head  of 
one  of  the  long  bones  is  removed  from  the  recently 
killed  animal,  split  into  two  down  the  middle  with 
a  strong  knife  or  a  chisel,  and  the  halves  placed  in 
a  large  quantity  of  a  solution  of  chromic  acid  of  the 
strength  above  indicated,  and  allowed  to  remain  in 
it  a  few  days.  The  exact  time  is  immaterial,  but  in 


92  PRACTICAL    HISTOLOGY. 

specimens,  which  are  left  rather  longer  in  the  liquid, 
the  bone  in  the  neighborhood  of  the  cartilage  is 
softened,  and  the  cartilage-cells  are  more  colored  by 
the  acid,  and  consequently  more  apparent.  When 
it  is  desired  to  prepare  the  tissue  for  the  microscope, 
one  of  the  halves  is  taken  from  the  fluid,  washed  in 
water  for  a  minute  or  two,  and  then,  the  bone  being 
held  in  the  hand,  one  or  two  thin  sections  are  taken 
from  the  surface  of  cleavage  vertically  to  the  articu- 
lar surface  and  extending  through  the  whole  depth 
of  the  cartilage,  including,  if  possible,  a  little  of  the 
adjacent  bone.  The  sections  are  placed  in  water  on 
a  slide  and  covered,  and  are  examined,  first  with  a 
low  power  to  see  the  general  arrangement  of  the 
cartilage  cell  groups  in  the  superficial,  intermediate 
and  deep  strata  respectively,  and  subsequently  with 
a  high  power  to  see  the  intimate  structure  of  the 
cells.  These  should,  as  before  indicated,  present  as 
nearly  as  possible  the  same  appearance  as  during  life, 
the  only  difference  being  that  the  tissue  generally  is 
less  transparent,  and  slightly  colored,  and  that  the 
cell-outlines  are  rather  more  strongly  marked.  These 
differences  become  even  less  obvious  when  glycerine 
has  been  permitted  to  diffuse  itself  under  the  cover- 
glass,  for  the  preservation  of  the  specimen. 

Sections  are  next  to  be  taken  parallel  to  the  articu- 
lar surface  and  mounted  in  the  same  manner;  but 
it  must  be  borne  in  mind  that  it  is  only  those  sec- 
tions which  include  parts  of  the  cartilage  near  the 
natural  or  artificial  surface  which  will  be  of  value 
as  respects  the  preservation  of  the  tissue  elements  in 
their  natural  condition,  at  least  in  the  case  of  thick 
articular  cartilages  of  large  animals.  For  the  preser- 
vative solution  naturally  takes  some  time  to  perme- 
ate the  cartilaginous  matrix,  and  before  it  has  time 
to  penetrate  to  the  deeper  parts,  the  cells  will  have 
already  shrunk  away  from  the  walls  of  the  inclosing 
cavities  and  have  become  changed  in  the  manner 
previously  indicated.  So  that  the  deeper  sections 
will  exhibit  merely  the  irregular,  contracted,  and 


CELL-SPACES    OF    CARTILAGE.  93 

highly  refracting  corpuscles  lying  loosely  in  their 
cell-spaces. 

At  the  thinnest  parts  of  all  the  sections  cavities 
may  he  observed  in  the  matrix  which  are  devoid  of 
cartilage-cells;  these  having  dropped  out  in  the  pro- 
cess of  preparation. 

Preparation  4. — These  cavities  or  cell-spaces  of 
cartilage  may  he  also  demonstrated  by  the  same 
method  as  was  employed  to  show  the  cell-spaces  of 
connective  tissue,  viz.,  treatment  with  nitrate  of 
silver  and  subsequent  exposure  to  the  light.  For 
this  purpose  a  fresh  joint  should  be  opened,  and  the 
articular  end  of  one  of  the  bones  (preferably  a  convex 
one)  removed  with  the  sawr  or  bone-forceps.  The 
end  thus  removed  is  rinsed  in  distilled  water  and 
then  quickly  transferred  to  nitrate  of  silver  solution 
(J  per  cent.),  in  which  it  is  allowed  to  remain  three 
minutes.  It  is  then  again  rinsed  in  water,  and  if 
necessary  gently  brushed  with  a  camel-hair  pencil 
to  remove  adhering  silver  precipitates  ;  after  which 
it  is  placed  in  a  beaker  of  weak  spirit  and  exposed 
to  sunlight.  "When  thoroughly  browned,  sections 
are  made  from  the  surface  with  a  razor  wetted  with 
spirit,  and  are  placed  in  water  (care  being  taken  that 
they  become  completely  immersed),  after  which  they 
may  be  mounted  in  glycerine. 

Some  of  the  sections  should  be  taken  from  near 
the  edge  of  the  cartilage  and  mounted  on  a  separate 
slide.  These  should  show7  the  branched  cell-spaces, 
which  present  a  transition  to  the  much  more  ramified 
spaces  of  the  connective  tissue  of  the  synovial  mem- 
brane. 

The  cells  themselves  which  occupy  the  spaces  are 
not  shown,  for,  as  in  silvered  tissues  generally,  the 
cell-protoplasm  remains  absolutely  unstained ;  indeed, 
it  is  at  first  sight  difficult  to  believe  that  the  rounded 
cavities  which  are  seen  really  contain  cartilage- cells. 
But  their  nuclei  may  be  brought  into  viewr  by  stain- 
ing with  logwood,  subsequently  the  matrix  being 
colored  by  the  nitrate  of  silver.  As  before  men- 


94  PRACTICAL    HISTOLOGY. 

tioned  in  speaking  of  the  connective  tissue,  it  is, 
however,  difficult,  if  not  impossible,  at  the  same  time 
to  bring  distinctly  into  view  the  outlines  of  the  cell- 
spaces  by  the  silver  method,  and  the  bodies  of  the 
cells  themselves  by  some  other  method  of  staining. 

Preparation  5. — No  doubt  one  of  the  best  and 
most  generally  useful  methods  of  coloring  cell  pro- 
toplasm is  that  known  as  the  gold  method.  This  is 
applied  to  cartilage  in  the  following  way.  Thin 
sections  are  made  from  the  articular  end  of  a  fresh 
bone  and  are  placed  in  a  few  drops  of  a  solution  of 
chloride  of  gold  (1  part  in  200  of  \vater).  After 
half  an  hour  they  are  transferred  to  a  beaker  con- 
taining a  comparatively  large  amount  of  distilled 
water  which  has  previously  been  slightly  acidulated 
with  acetic  acid,  just  so  as  to  be  distinctly  acid  to 
the  taste.  The  beaker  is  then  covered  with  a  glass 
plate  and  placed  in  a  window  in  as  warm  a  place  as 
possible,  and  where  it  will  be  exposed  for  some  hours 
a  day  to  the  full  sunlight.  Here  the  sections  are 
left  for  two  days, after  which  time  they  should  have 
acquired  a  dark  violet  color,  and  are  ready  to  mount 
in  glycerine.  But  before  this  is  done  they  should 
first  be  examined  with  a  low  power  in  a  drop  of 
water  to  see  whether  there  is  any  precipitated  matter 
upon  the  surfaces  of  the  sections.  If  this  is  the  case 
it  must  be  brushed  away  with  a  camel-hair  pencil, 
the  sections  being  held  with  fine  forceps  during  the 
process.  When  finally  mounted  and  examined  with 
a  high  power,  the  cartilage  cells  should  retain  pre- 
cisely their  natural  form  and  appearance, except  that 
they  are  stained  of  a  faint  violet  tinge,  whilst  the 
matrix  remains  almost  entirely  colorless.  In  sections 
taken  from  the  edge  of  the  curtilage,  the  ramified 
transitional  cells  which  occupy  the  corresponding 
cell-spaces  shown  by  the  silver  method  will  be  ren- 
dered apparent. 

The  coloration  by  the  gold  and  silver  methods  appears 
in  some  way  to  depend  on  the  occurrence  of  a  deposition 


GOLD    METHOD. 


95 


of  the  metal  in  those  parts  of  the  tissue  for  which  it  has 
the  greatest  affinity  ;  in  the  case  of  gold  this  is  generally 
the  protoplasm  of  the  cells  (and  the  nerve-fibres,  where 
any  exist) ;  in  the  case  of  silver  it  is  the  ground  substance, 
or  matrix,  or  intercellular  substance ;  so  that  the  results 
of  the  two  processes  may,  at  least  in  the  case  of  the  carti- 
lage-cells, be  looked  upon  as  standing  to  each  other  in  the 
light  of  a  positive  and  negative  image,  using  the  terms  as 
they  are  employed  in  photography. 

Fig.  19. 


Warming  nppnrntns  for  maintaining  portions  of  tissue,  after  treatment 
by  the  gold  method,  at  a  constant,  raised  temperature.  The  lower 
part  of  the  apparatus  is  filled  with  water,  into  which  the  bulb  of  the 
mercurial  regulator  dips. 

If  it  is  the  winter  time,  and  especially  if  there  is  very 
little  bright  sunlight,  it  is  of  the  greatest  importance  to 


96 


PRACTICAL    HISTOLOGY. 


keep  the  beaker  of  acidulated  water  in  which  the  tissue 
is  placed  warm,  as  this  materially  facilitates  the  reduc- 
tion of  the  gold.  For  this  purpose  it  is  well  to  have  some 
arrangement  by  which  the  beaker,  or  several  of  them,  if 
there  be  much  of  the  same  sort  of  work  going  on,  can  be 
kept  at  a  temperature  of  about  30°  or  40°  Centigrade, 
and  at  the  same  time  freely  exposed  to  the  light.  Fig. 
19  represents  a  convenient  apparatus  for  this  purpose. 
It  is  made  of  zinc  or  tinned  iron  with  loose  glass  sides 
and  top,  and  may  be  fitted  with  a  gas  regulator  (Fig.  20) 
to  prevent  the  temperature  from  rising  too  high.  When 
used  for  the  present  purpose  the  apparatus  should  of 
course  stand  in  a  good  light — in  the  window  if  possible. 


n,  a,  small  iuuer  tube,  capable  of  being  slipped  up 
or  down  withia  c,  the  upper  part  of  the  bulbed 
tube  containing  mercury,  d,  small  aperture  to 
allow  of  the  constant  passage  of  a  small  amount  of 
gas,  so  that  the  flame  is  not  entirely  extinguished 
if  the  mercury  should  rise  sufficiently  high  to 
entirely  exclude  the  lower  end  of  the  tube  a. 
The  tube  b,  with  the  corks  d  nod  e,  form  a  simple 
and  easily  constructed  arrangement  (suggested  by 
Mr.  F.  J.  M  Page)  which  serves  as  a  gas-tight 
telescopic  joiat,  and  enables  the  tube  a  to  be 
raised  or  lowered  according  as  the  temperature  of 
the  chamber  is  desired  to  be  higher  or  lower.  In 
all  other  respects  the  working  of  the  regulator  is 
similar  to  that  belonging  to  the  apparatus  shown 
in  Fig.  9. 


The  upper  part  of  the  raercural  gas-regulator  of  the  apparatus  shown  in 
Fig.  19.     Natural  size. 

Preparations  6  and  7.   Costal  Cartilage.— The 

cartilages  of  the  ribs  and  those  of  the  trachea  and 
larynx  may  be  prepared  and  examined  in  a  manner 
similar  to  that  recommended  for  the  study  of  articu- 
lar cartilage.  Sections,  both  parallel  and  vertical 
to  the  surface,  should  be  made  from  the  fresh  tissue 
and  from  pieces  which  have  been  a  longer  or  shorter 


CELL   TERRITORIES    OF    CARTILAGE.          97 

time  in  chromic  acid  solution  ;  those  from  the  latter 
being  mounted  and  preserved  in  glycerine  as  before. 

Preparation  8. — The  alum  process  may  also  be 
advantageously  used.  Sections,  as  thin  as  possible, 
of  a  perfectly  fresh  rib  cartilage  are  to  be  mounted 
in  a  dilute  solution  of  alum  (j  per  cent.).  This  will 
be  found  to  preserve  for  a  time  the  natural  appear- 
ance of  the  cells,  better  almost  than  any  other 
reagent. 

Preparation  9.  Cell  Territories  of  the  Matrix. 
— Sections  are  to  be  made  of  a  piece  of  thyroid 
cartilage  that  has  been  preserved  in  spirit,  and  are 
to  be  stained  with  logwood.  When  sufficiently 
colored,  they  are  transferred  to  water  and  then 
mounted  in  glycerine.  The  logwood,  besides  stain- 
ing the  nuclei  of  the  cells,  gives  the  matrix  also  a 
deep  purple  color.  But  this  coloration  of  the  matrix 
is  not  uniform,  for  some  parts  become  stained  much 
more  deeply  than  others;  those  regions  more  imme- 
diately around  the  cells  and  cell-groups,  and  which 
therefore  are,  as  commonly  considered,  the  latest 
formed  portions,  having  apparently  more  affinity 
for  the  coloring  matter  of  logwood  than  the  other 
and  older  parts.  By  this  method  the  whole  matrix 
appears  marked  out  into  what  may  be  termed  cell- 
territories^  although  series  of  definite  rings  can  by  no 
means  be  said  to  be  very  apparent  around  the  cell- 
groups,  such  as,  it  is  stated,  may  be  produced  in 
sections  of  cartilage  by  treating  them  with  a  mix- 
ture of  nitric  acid  and  chlorate  of  potash. 

Preparation  10.  Transition  to  yellow  fibro- 
cartilage, — If  the  arytenoid  cartilage  of  the  ox  is 
sliced  longitudinally,  it  exhibits  to  the  naked  eye  in 
its  lower  part  the  opaline  bluish  appearance  of  hya- 
line cartilage,  but  in  its  upper  part  the  faintly  yellow 
aspect  of  elastic  cartilage,  the  two  parts  being  sepa- 
rated by  a  distinct  line  of  demarcation.  Take  now  a 
thin  section  from  the  cut  surface,  including  a  little 
of  both  parts,  and  mount,  if  fresh,  in  alum  solution, 
or,  if  the  cartilage  have  been  previously  hardened  in 
9 


93  PRACTICAL    HISTOLOGY. 

spirit,  at  once  in  glycerine.  It  is  immediately  seen 
that  the  cartilage  above  the  line  of  demarcation 
merely  differs  from  that  below  by  the  superaddition 
of  a  network  of  comparatively  coarse  branching 
(elastic)  fibres.  Just  at  the  junction  of  the  two 
parts  the  fibres  are  but  few  in  number,  and  at  their 
ends  often  imperfect — i.  e.,  they  may  appear  continued 
merely  as  rows  of  granules  as  if  not  yet  fully  formed. 
Further  into  the  yellowish  part  they  permeate  the 
matrix  very  thickly,  and  give  it  almost  a  granular 
appearance,  owing  to  many  of  them  being  seen  in 
section.  But  immediately  around  each  cell  there  is 
always  a  larger  or  smaller  area  of  the  matrix  entirely 
free  from  fibres,  and  this  area  is  in  appearance  ex- 
actly like  the  matrix  of  hyaline  cartilage.  So  that 
we  may  conclude  that  yellow  or  elastic  cartilage  is 
merely  to  be  regarded  as  hyaline  cartilage,  in  the 
matrix  of  which  a  deposit  of  elastic  substance  has 
taken  place  in  the  form  of  branching  fibres;  and, 
indeed,  the  study  of  its  development  shows  this  to 
be  the  case. 

Preparation  11.  Yellow  fibre-cartilage. — A 
tissue  may  now  be  studied  which  is  composed  en- 
tirely of  yellow  cartilage,  the  human  epiglottis  or 
external  ear,  for  example.  A  section  of  either  of 
these  shows  what  at  first  looks  like  a  granular 
matrix  dotted  with  islands  of  hyaline  substance, 
each  with  one  or  two  cartilage-cells  in  the  centre. 
The  granular  appearance  of  the  matrix,  when  care- 
fully observed,  is  seen  to  be  due  to  an  excessively 
close  feltwork  of  fine  elastic  fibres;  in  fact,  the 
structure  is  quite  like  that  of  the  upper  part  of  the 
ox's  arytenoid,  although,  owing  to  their  fineness  and 
closeness,  the  individual  fibres  cannot  be  followed 
for  any  distance. 

Preparation  12.  White  fibro-cartilage  is  best 
studied  in  sections  of  any  ligament  which  is  com- 
posed of  this  tissue,  or  of  the  insertion  of  any  tendon 
or  ligament  which  is  attached,  as  in  the  young  sub- 
ject many  are,  to  cartilage.  The  tissue  may  have 


CELL-TERRITORIES    OF    CARTILAGE.          99 

been  previously  hardened  in  spirit.  Sections  should 
be  made  both  parallel  with  and  across  the  bundles 
of  tendinous  fibres.  They  are  to  be  stained  with 
logwood  and  mounted  in  glycerine.  It  will  be  seen 
in  the  longitudinal  sections  that  the  cartilage-cells 
lie  in  chains  between  the  tendon  bundles,  occupying 
the  place  of  the  ordinary  tendon-cells;  from  these 
they  are  chiefly  characterized  by  their  sharpness  of 
outline  and  (in  transverse  sections)  the  absence  of 
lamellar  extensions,  as  well  as  their  greater  thick- 
ness ;  but  transitions  between  the  two  are  not  unfre- 
quent,  especially  near  the  tendon  insertion. 


100  PRACTICAL    HISTOLOGY. 


CHAPTER    V. 

BONE. 

Preparations  1  and  2.  Transverse  and  longi- 
tudinal sections  of  hard  bone. — These  are  first 
cut  as  thin  as  possible  with  a  fine  saw,  and  are 
afterwards  reduced  in  thickness  and  polished  by 
grinding.  The  bone  selected  should  be  thoroughly 
macerated  and  bleached ;  it  should  be  absolutely 
free  from  grease.  Sections  may  be  made  from  dif- 
ferent bones,  flat  and  long,  but  for  typical  specimens 
of  the  compact  tissue,  transverse  and  longitudinal 
sections  of  one  of  the  long  bones  of  the  limbs — the 
ulna,  for  instance — may  be  recommended,  and  a 
vertical  section  of  one  of  the  flat  bones  of  the  skull, 
such  as  the  parietal,  should  also  be  prepared. 

The  first  thing  to  be  done  is  to  get  as  thin  a 
piece  as  possible  cut  with  a  saw  from  any  desired 
part.  For  the  purpose  a  fret-saw  may  be  used, 
unless  a  circular  saw  is  available,  when  this,  if  fine 
enough,  may  be  employed  with  advantage.  The 
piece  so  obtained  is  ground  down  on  a  hone  wetted 
with  water.  The  hone  must  have  been  previously 
freed  from  all  traces  of  greasy  matter  by  washing 
with  soap  and  water  with  a  little  soda.  The  piece 
of  bone  is  pressed  down  and  rubbed  to  and  fro  on 
the  hone  simply  by  the  finger,  being  ground  first 
on  one  side  and  then  on  the  other.  The  feel  will 
be  almost  sufficient  to  tell  when  it  is  thin  enough, 
and  this  may  be  confirmed  by  placing  it  on  a  slide 
without  covering  it  and  examining  with  a  low 
power.  Numerous  scratches  will  doubtless  be  visi- 
ble on  its  surface,  produced  by  the  grain  or'  the  stone, 
however  fine  this  may  be  ;  but  unless  very  obvious, 


SECTIONS    OF    HARDENED    BONE.  101 

they  may  be  disregarded,  for  they  become  almost 
invisible  in  the  subsequent  preparation.  This  con- 
sists in  well  rinsing  the  piece  (when  satisfied  that  it 
is  thin  enough)  in  clean  water  by  aid  of  a  hair 
pencil,  placing  it  upon  a  slide  to  dry,  and,  when 
dry,  mounting  it  in  Canada  balsam.  The  balsam 
to  be  used  for  this  purpose  must  not  be,  as  is  usually 
the  case  with  that  sold  in  the  shops,  semi-fluid,  but 
quite  hard  in  the  cold — a  condition  which  results 
from  long  keeping,  or  may  be  produced  by  heating  a 
little  of  the  more  recent  resin  in  a  capsule  over  a 
sand-  or  wTater-bath,  until  all  the  volatile  matters  are 
driven  off.  A  drop  of  the  melted  balsam  is  then 
placed  upon  a  slide^  which  is  warmed  over  a  flame 
until  the  resin  has  diffused  itself  pretty  evenly  over 
the  central  part  of  the  slide.  This  is  then  placed 
on  the  table,  and  whilst  cooling,  but  before  the 
balsam  has  time  to  become  quite  hard  again,  the 
thin  piece  of  bone  is  placed  upon  it.  A  clean  cover- 
glass  is  then  taken  up  by  the  forceps,  a  drop  of  the 
balsam  placed  upon  it  also  warmed  in  like  manner, 
and  quickly  inverted  over  the  preparation.  By 
this  mode  of  proceeding  the  balsam  fills  up  and 
renders  invisible  the  scratches  on  the  surfaces  of  the 
section  of  bone,  and  some  of  the  Haversian  canals 
may  also  be  filled  by  it ;  but  it  becomes  solidified 
before  it  has  time  to  penetrate  into  the  lacunae  and 
canaliculi,  which  remain,  therefore,  filled  with  air, 
and  present  the  black  appearance  which  is  char- 
acteristic of  any  small  cavities  containing  air  when 
they  are  viewed  under  the  microscope  by  trans- 
mitted light. 

Most  of  the  structural  points  with  regard  to  bone 
can  be  seen  much  better  in  these  preparations  of  the 
hard  tissue  than  in  sections  of  a  decalcified  bone. 
But  it  is  important  in  mounting  the  pieces  after 
they  have  been  ground  down  to  be  careful  that  the 
balsam  does  not  remain  fluid  long  enough  to  have 
time  to  penetrate  into  the  thickness  of  the  section  ; 
if  this  should  happen,  the  whole  is  rendered  too 

9* 


102  PRACTICAL    HISTOLOGY. 

transparent  for  any  of  the  details  of  structure  to  be 
made  out.  On  the  whole  it  is  perhaps  advisable  to 
purchase  one  or  two  good  specimens  rather  than 
devote  a  large  amount  of  time  to  the  manufacture 
of  what  may  after  all  turn  out  to  be  but  an  indif- 
ferent preparation.  Sections  of  bard  bone  and  of 
teeth  are  amongst  the  very  few  histological  prepara- 
tions which  are  usually  better  made  by  those  who 
make  a  business  of  preparing,  microscopic  specimens 
for  sale  than  by  the  student  himself. 

Preparation  3. — In  addition  to  sections  of  hard 
bone  which  are  made  by  grinding  in  the  manner 
above  described,  a  portion  of  a  well-macerated  bone 
may  be  placed  in  a  solution  of  hydrochloric  acid 
(10  parts  of  the  commercial  acid  to  100  of  water) 
until  all  the  earthy  matter  is  dissolved  ;  the  piece  is 
then  steeped  in  a  very  weak  solution  of  carbonate 
of  soda  to  free  it  from  the  remains  of  the  acid,  and 
sections  may  then  be  made  with  a  razor  or  sharp 
scalpel  and  mounted  in  glycerine.  But  these  decal- 
cified specimens  in  no  way  illustrate  the  structure 
of  the  tissue  so  well  as  the  others. 

Preparation  4. — It  is,  however,  quite  another 
question  with  regard  to  bones  which  have  not  been 
submitted  to  previous  maceration.  For  it  is  to  be 
remembered  that  in  considering  the  structure  of  a 
bone  we  have  to  deal  not  merely  with  the  bony 
matter  pure  and  simple  and  its  included  cavities, 
but  that  there  are  in  addition  the  soft  contents  of 
those  cavities — the  corpuscles  in  the  lacunae,  the 
bloodvessels  in  the  Haversian  canals,  and  the  marrow 
in  the  medullary  cavity  and  in  the  interspaces  of  the 
spongy  tissue.  In  order  properly  to  view  these 
perishable  structures,  we  must  employ  a  reagent 
which  will  at  the  same  time  decalcify  and  soften 
the  hard  matter  and  preserve  and  harden  the  in- 
cluded soft  tissues.  Such  a  reagent  is  to  be  found 
in  a  solution  of  chromic  acid,  but  ns  the  decalcify- 
ing powers  of  this  are  but  feeble,  the  portions  of 
bone  placed  in  it  must  be  as  small  as  possible,  and  a 


SECTIONS    OF    SOFTENED    BONE.  103 

large  proportionate  amount  of  the  fluid  must  be 
used.  For  example  if  the  bone  be  a  long  bone 
about  the  size  of  the  human  metacarpal,  a  piece  not 
longer  than  a  quarter  of  an  inch  should  be  sawn 
from  it  and  placed  (suspended  by  a  thread  if  pos- 
sible) in  a  beaker  capable  of  containing  some  200 
c.  c.  of  the  chromic  acid  solution.  If  the  bone  be 
larger,  it  is  well  to  split  the  disk  thus  sawn  off  into 
three  or  four  smaller  pieces,  since  otherwise  the 
decalcification  will  occupy  too  long  a  time.  The 
acid  used  should  be  at  first  very  weak  (1  in  600) ; 
in  two  days'  time  this  may  be  changed  for  a  solu- 
tion of  1  in  400  ;  and  this  again  in  another  two  days 
for  1  in  200.  Beyond  this  the  strength  of  the  acid 
should  not  be  increased,  but  the  fluid  should  be 
renewed  every  three  days  at  least.  In  addition  to 
this  frequent  changing  of  the  liquid,  it  should, 
throughout  the  whole  time  of  softening,  be  stirred 
as  often  as  possible:  this  is  of  the  greatest  impor- 
tance, for  every  agitation  brings  fresh  portions  of 
acid  to  attack  the  earthy  matter  of  the  bone.  By 
attention  to  this  particular  the  time  which  the 
pieces  take  to  become  thoroughly  decalcified  may 
be  materially  shortened.  The  completion  of  the' 
process  is  ascertained  by  passing  a  needle  through 
the  middle  of  the  piece  employed  ;  if  it  meets  with 
no  gritty  obstruction,  all  the  earthy  matter  is  pro- 
bably got  rid  of.  This  will  most  likely  be  the  case 
in  two  or  three  weeks  from  the  commencement  of 
the  operation.  Should  the  pieces  of  bone,  owing  to 
their  too  great  thickness  or  density,  be  much  longer 
than  this" before  they  are  sufficiently  soft,  the  pro-. 
cess  may  be  hastened  by  adding  to  the  chromic  acid 
solution  a  little  nitric  acid  (2  c.  c.  to  each  100  c.  c. 
of  chromic  solution).  This  reagent  was  not  avail- 
able at  the  commencement  of  the  operation,  because, 
although  it  would  have  more  readily  dissolved  the 
salts  of  lime  than  the  chromic  acid,  yet  it  would 
have  softened  still  more  the  included  soft  tissues  of 
the  bone  instead  of  hardening;  them  like  that  re- 


104:  PRACTICAL    HISTOLOGY. 

agent ;  but  it  may  be  employed  during  the  later 
stages,  especially  in  conjunction  with  the  chromic 
acid,  since  the  latter  reagent  has,  by  its  peculiar 
coagulant  action  upon  the  animal  tissues,  so  altered 
their  constitution  that  their  structure  is  no  longer 
rendered  indistinct  by  diluted  nitric,  acetic,  and 
the  ordinary  acids,  which  produce  swelling  and 
maceration  of  the  fresh  tissues,  and  especially  of  the 
connective  tissue. 

The  process  of  decalcification  being  completed,  the 
pieces  are  placed  in  water  for  a  few  hours  to  get  rid 
of  the  excess  of  chromic  acid  imbibed,  and  are  then 
transferred  to  spirit;  or  sections  may  be  made  at  once 
without  placing  the  piece  in  spirit  at  all.  In  cutting 
them,  if  the  bit  of  bone  is  too  small  to  be  held  in  the 
fingers,  it  may  be  placed  in  a  split  piece  of  cork.  The 
sections  should  be  very  thin,  but  it  is  not  necessary 
that  they  should  be  large  or  include  the  whole  thick- 
ness of  the  bone;  they  are  to  be  stained  in  the  log- 
wood solution,  washed  in  water,  and  finally  mounted 
in  glycerine.  The  corpuscles  within  the  lacunae,  or 
at  all  events  their  nuclei,  are  beautifully  shown,  and 
all  the  soft  parts  are  more  or  less  stained,  but  the 
actual  substance  of  the  bone  is  very  slightly  colored. 
At  the  same  time,  any  lines  of  demarcation  which 
may  be  present  indicating  successive  deposits  of 
osseous  matter  in  the  formation  of  the  bone,  or 
absorption  at  any  part  and  subsequent  redisposition, 
with  the  characteristic  scalloped  edge  which  such  a 
junction  almost  always  possesses,  are  very  clearly 
shown  by  a  difference  in  the  coloration.  Moreover, 
if  in  the  section  there  happens  to  be  any  portion 
remaining  of  the  (ossified)  matrix  of  the  original 
embryonic  cartilage,  this,  like  cartilage  matrix  gene- 
rally, is  intensely  "stained  by  the  logwood. 

Preparation  5. — Instead  of  chromic,  picric  acid 
may  be  used  for  the  decalcification.  A  saturated 
solution  of  the  acid  is  employed,  and  care  is  to  be 
taken  constantly  to  supply  fresh  crystals  of  picric 
acid  to  take  the  place  of  that  which  is  used  up  in 


PICRIC    ACID    METHOD.  105 

dissolving  the  lime  salts.  In  all  other  respects  the 
proceeding  is  much  the  same  as  for  the  chromic  acid 
method,  except  that  the  pieces  will  require  much 
longer  washing  in  water,  and  that  the  spirit  in  which 
they  are  placed  be  changed  several  times  before  the 
excess  of  picric  acid  can  be  got  rid  of.  The  sections 
may  be  stained  with  logwood  and  mounted  in  glyce- 
rine in  a  similar  manner,  and  will  show  the  structure 
as  well,  if  not  better,  than  those  prepared  with 
chromic  acid. 

Preparation  6.  Lamellae  and  Sharpey's  per- 
forating fibres. — For  showing  these  structures,  ma- 
cerated bones  that  have  been  decalcified  by  hydro- 
chloric acid  serve  very  well.  They  should  before 
use  soak  in  water  for  some  time,  and  subsequently 
lie  in  spirit,  so  as  to  get  rid  of  the  last  traces  of  the 
acid.  The  point  of  one  of  the  blades  of  a  sharp  pair 
of  forceps  is  then  inserted  obliquely  into  the  bone  at 
its  outer  surface,  and  a  small  piece  is  gripped  by  the 
other  blade  and  dragged  off  in  such  a  manner  that  it 
pulls  away  with  it  a  very  thin  strip  from  the  super- 
ficies of  the  bone.  A  few  such  strips  having  been 
obtained,  either  from  different  parts  of  the  bone  or 
from  the  same  place  (at  different  depths,  therefore), 
they  are  placed  in  water  on  a  slide  with  the  inner 
surface  uppermost  and  examined  with  a  low  power. 
In  some  places  tapering  fibres  will  be  seen  projecting 
from  the  surface  of  the  torn-off  strip  like  nails  pro- 
jecting from  a  board ;  in  other  parts,  round  or  ovalish 
holes,  corresponding  generally  in  size  with  these 
fibres,  will  be  apparent;  these  are  apertures  in  the 
lamellae  where  the  latter  have  been  pierced  by  the 
fibres  of  Sharpey,  but  those  fibres  have  been  pulled 
out  in  tearing  off  the  strips.  Further,  there  may  be 
made  out  a  faint  appearance  of  decussation  in  the 
lamellae,  like  the  checks  on  a  plaid,  but  oblique  in 
direction,  best  marked  near  the  thin  edge  of  the  strip 
of  bone.  These  appearances  are  all  more  evident 
when  the  preparation  is  covered  and  examined  with 
a  higher  power.  To  preserve  the  preparation  it  is  not 


106  PRACTICAL    HISTOLOGY. 

a  good  plan  to  replace  the  water  in  which  the  strips 
are  mounted  by  glycerine,  since  this  renders  the  tex- 
ture too  transparent  and  the  outlines  too  indefinite. 
But  the  strips  may  be  mounted  and  preserved  for  a 
considerable  time  in  water  merely  if  the  edges  of  the 
cover-glass  are  fixed  first  by  melted  paraffin,  and  then 
by  layer  after  layer  of  chloroform  balsam,  so  as  to 
obviate  as  much  as  may  be  the  risk  of  evaporation. 

The  bones  in  which  the  fibres  of  Sharpey  may  best  be 
demonstrated  in  the  way  above  described  are  the  flat 
bones  of  the  skull.  They  are  also  to  be  seen  in  similar 
preparations,  and  in  sections  of  the  long  bones.  If  a  sec- 
tion be  made  of  such  a  bone  at  the  place  of  insertion  into 
it  of  a  tendon  or  ligament,  and  in  the  direction  of  the  fibres 
of  the  tendon  or  ligament,  it  will  be  seen  that  the  bundles 
of  fibres  of  the  last-named  structures  are  continued  into 
the  substance  of  the  bone  as  perforating  fibres  or  fibres  of 
Sharpey,  so  that  these  in  fact  almost  compose  the  whole  of 
the  osseous  tissue  at  this  place  (Ranvier).  This  shows, 
moreover,  that  the  fibres  of  Sharpey  are  to  be  regarded  as 
bundles  of  fibrous  tissue  (connected  either  with  the  perios- 
teum or  with  a  tendon  or  ligament)  which  were  intercalated 
with  the  osseous  substance  proper  when  this  was  formed 
and  have  become  ossified  at  the  same  time.  When  tendons 
undergo  ossification,  the  bony  substance  which  is  formed 
is  wholly  of  the  same  nature  as  the  fibres  of  Sharpey; 
this  may  be  characteristically  seen  in  the  ossified  tendons 
which  are  met  with  in  the  legs  of  birds. 

Preparation  7.  Development  of  bone  in 
membrane. — For  the  study  of  the  intra-membra- 
nous  process  of  .ossification  it  is  best  to  employ  the 
flat  bones  of  the  skull  of  sheep's  embryos  from  two 
to  three  inches  long.  The  embryos  may  have  been 
preserved  in  Muller's  fluid,1  or  spirit,  or  they  may 
be  employed  fresh.  A  piece  (corresponding  in  posi- 
tion with  the  future  parietal,  for  example)  of  the 
still  membranous  skull-cap  is  cut  out  with  fine  scis- 
sors, placed  under  water  if  from  Muller's  fluid,  under 

1  A  solution  containing  2£  parts  bichromate  of  potash,  and  1 
part  sulphate  of  soda,  to  100  of  water. 


PROCESS    OF    OSSIFICATION.  107 

salt  solution  if  recent,  and  the  skin  and  muscular 
layers  are  torn  away  from  the  outside  and  the  dura 
mater  and  cartilaginous  layer  (which  in  these  animals 
rises  up  laterally  from  the  cartilaginous  hasis  cranii) 
from  the  inside.  The  membrane  in  which  the  bone 
is  being  formed  is  then  left.  It  is  held  upon  a  glass 
slide  with  a  needle  or  line  forceps,  and  brushed 
firmly  with  a  camel-hair  pencil,  the  hairs  of  which 
have  been  cut  off  short  so  as  to  render  the  stump 
stiff  and  resisting.  The  piece  must  be  kept  wet, 
and  examined  from  time  to  time  with  a  low  power 
to  see  whether  the  edges  of  the  newly-formed  bone 
are  sufficiently  clear  of  the  membrane  and  corpuscles, 
so  that  the  osseous  spicules  and  their  fibrous  pro- 
longations are  readily  seen.  When  this  is  the  case 
the  piece  is  held  with  the  forceps  and  well  rinsed  in 
water  or  salt  solution  to  free  it  from  loose  particles 
of  the  soft  tissue ;  it  is  then  placed  in  logwood  solu- 
tion, and  when  sufficiently  colored  (two  or  three 
minutes)  is  mounted  in  glycerine.  The  osteoblasts 
will  be  found  stained  by  the  logwood ;  the  ossified 
part  is  dark  and  highly  refracting,  the  osteogenic 
fibres  by  which  it  is  prolonged  remain  clear  and 
colorless. 

The  process  of  intra-membranous  development 
may  also  be  very  advantageously  studied  in  sections 
of  the  lower  jaw  of  the  foetus  or  young  animal,  as 
will  be  afterwards  pointed  out  in  describing  the 
mode  of  preparing  this  to  show  the  development  of 
other  structures,  the  teeth  and  hair,  for  example. 

Preparation  8.  Development  of  bone  in 
cartilage, — To  study  the  mode  of  ossification  in 
cartilage  it  is  not  necessary  to  have  recourse  to  the 
bones  of  a  fcetal  animal,  since  any  which  are  still  in 
process  of  growth  will  serve  the  same  end,  and  it  is 
more  convenient  for  purposes  of  manipulation  if 
they  have  attained  a  certain  size.  The  long  bones 
of  a  new-born  kitten  may  advantageously  be  em- 
ployed, and  the  best  preparations  are  certainly  those 
obtained  from  the  recently  killed  animal,  although 


108  PRACTICAL    HISTOLOGY. 

for  permanent  preservation  it  is  better  that  they 
should  have  been  some  days  in  Mtiller's  fluid.  The 
mode  of  preparing  the  fresh  specimens  is  as  follows. 
One  of  the  long  bones — the  femur,  for  instance — is 
removed,  and,  its  cartilaginous  head  having  been 
cleared  of  the  adherent  soft  structures,  is  split  down 
the  middle  with  a  strong  scalpel,  the  split  extending 
a  little  distance  into  the  subjacent  bone.  Then  by 
a  movement  of  the  scalpel  one  of  the  halves  is  broken 
away,  and  the  junction  of  the  bone  and  cartilage  is 
brought  clearly  into  view.  One  or  two  slices  as  thin 
as  possible  are  now  taken  from  the  surface  thus  pro- 
duced, including  the  line  of  advancing  ossification, 
and  are  placed  on  a  slide  in  salt  solution  and  covered. 
These  sections  are  of  course  parallel  with  the  long 
axis  of  the  bone  ;  but  to  complete  the  observation 
others  should  be  made  across  that  axis,  at  and  a 
little  below  the  level  of  the  line  of  ossification,  the 
cartilaginous  head  being  first  sliced  gradually  away 
until  the  line  of  ossification  is  reached,  and  then  a, 
series  of  sections  taken  of  the  part  of  the  cartilage 
which  is  undergoing  ossification  and  of  the  newly- 
formed  bone. 

The  fresh  preparations  are  by  far  the  most  beau- 
tiful and  instructive  if  the  sections  are  obtained 
sufficiently  thin.  If  it  is  wished  permanently  to 
preserve  a  section  made  in  this  way,  the  salt  solution 
in  which  it  is  mounted  must  be  drawn  off  by  filter- 
ing paper  placed  at  one  edge  of  the  cover-glass,  and 
at  the  same  time  replaced  by  a  solution  of  osmic 
acid  (one  per  cent.)  applied  at  the  opposite  edge. 
The  preparation  may  then  be  left  for  an  hour  (the 
filter  paper  being  removed),  after  which  time  the 
osrnic  acid  is  in  its  turn  withdrawn  arid  replaced  by 
water,  and  finally  a  drop  of  glycerine  is  put  at  the 
edge  of  the  covering-glass  and  suffered  to  take  the 
place  of  the  water  as  this  evaporates. 

Preparation  9. — Sections,  longitudinal  and  trans- 
verse, of  ossifying  bones  which  have  been  in  Mliller's 
fluid  for  a  few  days,  are  prepared  in  exactly  the 


PROCESS    OF    OSSIFICATION.  109 

same  manner,  except  that  the  treatment  with  osmic 
acid  is  here  unnecessary.  The  preparations  can  be 
mounted  at  once  in  glycerine. 

Preparation  10. — To  trace  the  later  steps  of  the 
ossification,  it  is  requisite  to  obtain  sections  extending 
rather  more  deeply  into  the  newly-formed  bone  than  is 
possible  whilst  this  still  retains  its  earthy  salts.  For 
this  purpose,  therefore,  the  hone,  which  may  with  advan- 
tage be  considerably  smaller  than  that  of  the  new-born 
kitten — one  from  a  small  foatus,  for  example — is  decalci- 
fied in  chromic  or  picric  acid  in  the  same  way  as  was 
employed  for  the  deealcification  of  pieces  of  the  fully- 
developed  bone  (Preps.  4  and  5).  But  if  the  foetal  bone 
is  small,  the  time  necessary  for  such  decalcification  will 
be  very  much  shorter  than  was  requisite  for  the  dense 
adult  bone.  Jn  order  to  cut  a  longitudinal  section  suffi- 
ciently extensive  and  thin,  it  will  be  necessary  to  embed 
the  bone  in  the  wax  mass  (chap,  x.),  and  the  sections 
obtained  may  be  stained  with  logwood,  or  successively 
with  both  logwood  and  carmine,  and  mounted  in  dammar 
varnish  in  the  way  usually  employed  for  treating  sections. 
A  still  better  plan  is  to  stain  the  bone  entire  with  an 
alcoholic  solution  of  magenta,  allow  it  to  become  per- 
meated first  with  oil  of  cloves  and  then  with  melted 
cacao-butter,  and  afterwards  embed  in  cacao-butter,  treat- 
ing the  sections  with  warm  oil  of  cloves  to  dissolve  out 
the  fat,  and  then  mounting  in  dammar  varnish.  Or  the 
stained  bone  may  be  permeated  with  gurn,  placed  in  spirit 
containing  one-sixth  its  volume  of  water  to  harden  the 
gum  within  the  tissue,  embedded  in  wax  mass,  cut,  and 
the  sections  placed  in  water  to  dissolve  out  the  gum. 
The  .object  of  allowing  a  tissue  of  this  nature,  which  con- 
sists of  hard  and  soft  parts  intermingled,  to  become 
permeated  by  the  substance  above  recommended,  is  that 
the  soft  parts  and  cavities  may  be  filled  up  by  a  hard 
material,  so  that  the  tissue  may  offer  a  uniform  resistance 
to  the  razor  and  the  parts  of  a  section  may  cohere ; 
otherwise  the  soft  parts  are  very  apt  to  become  separated 
from  the  hard. 

But  since  these  processes  are  somewhat  complicated, 
the  student  is  recommended  to  defer  them  for  a  while 
10 


110  PRACTICAL    HISTOLOGY. 

until  the  methods  upon  which  they  depend   have  been 
described  and  explained. 

Preparation  11.  Medullary  tissue. — Ordinary 
marrow,  which  consists  for  the  most  part  of  adipose 
tissue,  is  obtained  from  the  long  bones  of  most 
animals.  But  the  spongy  tissue  of  the  bones  gene- 
rally and  the  medullary  canal  of  the  long  bones 
in  some  animals — the  rabbit  and  guinea-pig,  for 
example — are  filled  with  red  marrow,  which  con- 
tains little  adipose  tissue,  but  is  mainly  made  up 
of  the  so-called  proper  marrow-cells,  which  are  in 
many  respects  similar  to  the  pale  corpuscles  of  the 
blood,  and,  like  these,  exhibit  amoeboid  movements. 
To  see  the  marrow-cells,  therefore,  in  their  natural 
condition  the  tissue  should  be  taken  quite  fresh  and 
examined  on  the  warm  stage.  The  bone — the  femur 
of  a  guinea-pig,  for  instance,  preferably  a  young 
animal — having  been  removed  and  cleared  of  the 
surrounding  soft  parts,  is  broken  across,  and  a  small 
piece  of  the  marrow  picked  out  and  broken  up  with 
needles  in  a  drop  of  serum  or  salt  solution  on  a  piece 
of  thin  glass.  A  small  piece  of  hair  is  then  added, 
and  the  preparation  is  covered  ;  a  brush  dipped  in 
oil  is  drawn  round  the  edge  of  the  cover-glass  to 
prevent  evaporation,  and  the  specimen  is  then  placed 
on  the  wrarrn  stage  (p.  30)  and  examined.  The  dif- 
ferent sizes  and  varying  forms  of  the  marrow-cells 
are  to  be  noted,  as  also  the  large  clear  nucleus  which 
many  of  them  possess,  and  which  at  once  distin- 
guishes these  cells  from  the  ordinary  white  blood- 
corpuscles.  But  many  are  also  observable  which 
are  in  every  respect  similar  to  the  latter,  and  may 
indeed  not  improbably  belong  to  the  blood,  for  the 
bloodvessels  are  very  large  and  numerous  in  the 
medullary  tissue,  and  yield  the  numerous  red  blood- 
corpuscles  which  are  seen  scattered  over  the  field. 
Transitions  may  be  observed  between  the  proper 
marrow-cells  and  the  white  corpuscles,  and  as  they 
both  exhibit  amoeboid  movements  it  may  be  inferred 


MARROW.  Ill 

that  they  are  essentially  of  the  same  nature,  if  indeed 
the  one  is  not  to  be  regarded  as  derived  from  the 
other. 

Other  cells  may  perhaps  he  met  with  much  fewer 
in  number, flattened  in  form,  and  sometimes  branched, 
with  a  large  clear  oval  nucleus,  and  in  some  instances 
containing  yellowish -red  pigment  granules.  They 
are  larger  than  the  proper  marrow-cells,  and  exhibit 
no  changes  of  form.  They  appear  to  be  connective 
tissue-corpuscles.  There  is  yet  another  element  to 
be  found  in  the  marrow — most  likely  to  be  met  with 
in  a  bit  taken  from  near  the  inner  surface  of  the  bone. 
This  is  the  rnyeloplaque  or  ostoclast,  and  is  charac- 
terized by  its  enormous  size — whence  the  name  giant 
cell — by  its  granular  appearance,  and  by  containing 
a  number  of  clear  round  or  oval  nuclei  grouped 
together  in  the  middle  of  the  cell,  or,  in  some  cases, 
a  single  large  irregular  nucleus  with  numerous  buds 
from  its  circumference. 

Preparation  12. — To  isolate  these  various  ele- 
ments of  the  marrow  better  than  can  be  done  in  the 
fresh  condition,  a  piece  of  the  tissue  is  to  be  placed 
in  weak  spirit  (ordinary  methylated  spirit  diluted 
with  twice  its  bulk  of  water)  for  a  day  or  two. 
After  this  time  a  small  portion  is  thoroughly  broken 
up  with  needles  in  a  drop  of  water  on  a  slide,  is  then 
covered,  and  stained  by  allowing  dilute  logwood 
solution  to  flow  in  under  the  edge  of  the  cover-glass. 
The  logwood  is  replaced  in  a  minute  or  two  by  water, 
and  this  again  by  glycerine.  By  this  means  a  per- 
manent preparation  is  obtained,  which  can  be  studied 
at  leisure,  and  which  very  well  exhibits  the  different 
kinds  of  cells  above  enumerated,  whilst  the  red 
blood-corpuscles  are  rendered  almost  completely  in- 
visible. 


112  PRACTICAL    HISTOLOGY. 


CHAPTER    VI. 

MUSCULAR  TISSUE. 

Preparation  1.    Involuntary  muscle. — It  is 

very  easy  to  isolate  the  lanceolate  cells  of  which  this 
tissue  consists.  For  this  purpose  all  that  is  neces- 
sary is  to  place  a  piece  of  any  organ  containing  plain 
muscular  tissue — the  intestine,  for  instance — in  a 
weak  solution  of  bichromate  of  potash  (one  part  to 
800  of  water)  for  forty-eight  hours.  At  the  end  of 
this  period  of  maceration,  a  small  strip  of  either  the 
longitudinal  or  the  circular  muscular  fibres  is  torn 
off  with  the  forceps,  placed  in  a  drop  of  water  on  a 
slide,  and  separated  as  finely  as  possible  with  needles. 
A  cover-glass  is  then  laid  on,  and  the  preparation  is 
carefully  examined  with  a  high  power.  The  ends 
and  edges  of  the  larger  pieces  of  the  tissue  have  a 
somewhat  ragged  aspect,  due  to  the  projection  from 
them  of  the^  tapering  ends  of  the  fibre-cells.  In 
addition  to  these  partially  separated  elements,  others 
are  to  be  met  with  scattered  over  the  preparation 
which  are  wholly  free,  and  in  which  all  the  charac- 
teristic appearances  of  this  tissue  can  be  distinctly 
made  out.  The  elongated  nucleus  in  the  middle  of 
each  riband-shaped  cell  can  be  seen  in  those  cells 
which  lie  flat,  but  it  is  at  present  rather  indistinct. 
It  may,  however,  be  brought  more  clearly  into 
view,  as  can  also  the  faint  longitudinal  striation 
wliich  the  cells  exhibit,  by  employing  one  of  the 
smaller  holes  in  the  diaphragm  of  the  microscope  to 
admit  the  light  to  the  object.  But  to  show  clearly 
the  nuclei  ol^the  plain  muscular  fibre-cells,  nothing 
is  better  adapted  than  staining  the  tissue  with  a 
weak  solution  of  logwood-alum.  It  must  be  used 


INVOLUNTARY    MUSCLE.  113 

quite  dilute,  and  suffered  gradually  to  diffuse  itself 
under  the  cover-glass  from  a  small  drop  placed  at 
one  edge.  It  is  not  a  good  plan  to  draw  it  through 
by  means  of  blotting-paper,  since  in  this  way  many 
of  the  isolated  elements  of  the  tissue  will  be  drawn 
away  at  the  same  time.  But  after  the  drop  of  log- 
wood solution  has  passed  in  great  measure  or  entirely 
under  the  cover-glass,  a  small  drop  of  strong  glyce- 
rine may  be  added  at  the  same  spot  as  the  logwood. 
This  as  it  diffuses  under  the  cover  will  gradually 
push,  as  it  were,  the  logwood  solution  before  it,  so 
as  to  cause  the  staining  fluid  to  traverse  successively 
every  part  of  the  preparation,  and  eventually  to 
become  collected  entirely  at  the  opposite  edge,  the 
water  meanwhile  evaporating  and  leaving  the  glyce- 
rine in  possession  of  the  field.  All  that  is  needed 
to  complete  the  preparation  is,  in  the  course  of  a  day 
or  two,  to  fix  the  cover-glass  by  painting  a  little 
chloroform  balsam  around  the  edges. 

The  involuntary  muscular  fibres  will  be  seen  in 
section  in  preparations  of  the  stomach  and  intestine 
and  numerous  other  organs,  so  that  it  is  not  neces- 
sary to  make  special  preparations  at  this  stage  for 
the  purpose.  It  may,  however,  be  instructive  to 
demonstrate  the  manner  in  which  the  cells  are 
applied  edge  to  edge  in  order  to  make  up  the 
bundles  and  lamellae  of  the  tissue.  This  is  often 
shown  sufficiently  well  in  a  thin  strip  which  has 
been  stained  with  logwood  and  mounted  in  glyce- 
rine without  teasing. 

Preparation  2. — But  the  best  preparations  for 
exhibiting  the  arrangement  of  the  cells  are  those 
stained  with  nitrate  of  silver.  As  in  other  tissues, 
this  reagent  stains  only  the  intercellular  substance, 
leaving  the  cells  themselves  uncolored  ;  their  out- 
lines are  thus  brought  very  distinctly  into  view. 
The  way  to  prepare  the  muscular  coat  of  the  intes- 
tine by  this  method  is  as  follows :  A  piece  of  intes- 
tine is  removed  from  the  recently-killed  animal  (the 
large  intestine  of  the  frog  answers  verv  well  for  the 

10* 


114  PRACTICAL    HISTOLOGY. 

purpose),  and  one  end  being  tied  up,  a  glass  canula 
is  fastened  into  the  other  end,  and  the  piece  of 
intestines  having  been  distended  with  air,  this  also 
is  closed  by  a  ligature  and  the  canula  removed.  The 
outer  surface  is  then  brushed  vigorously  with  a 
camel-hair  pencil  wetted  with  distilled  water  to 
remove  the  epithelioid  cells  of  the  serous  membrane ; 
a  few  drops  of  a  solution  of  nitrate  of  silver  (i  per 
cent.)  are  allowed  to  flow  over  the  brushed  surface 
and  to  remain  in  contact  with  it  for  two  minutes, 
the  silver  solution  is  then  washed  off  by  a  stream  of 
distilled  water,  and  finally  the  distended  intestine 
is  immersed  in  a  mixture  of  equal  parts  of  water 
and  spirit  in  a  beaker  and  exposed  to  the  light.  The 
immersion  is  effected  either  by  tying  a  piece  of 
glass  rod  or  other  heavy  substance  to  the  gut,  or  by 
filling  the  beaker  to  the  brim  and  placing  a  glass 
plate  over  it  so  as  to  press  the  piece  of  intestine 
under  the  surface  of  the  fluid.  In  about  an  hour's 
time  the  preparation  may  be  removed  from  the 
light  (a  few  minutes  will  be  sufficient  if  it  is  bright 
sunlight),  and  a  small  piece  is  then  to  be  cut  out 
and  mounted  in  glycerine  with  the  outer  surface 
uppermost. 

Preparation  3.  Voluntary  muscular  tissue : 
cross-striped  muscle. — For  the  examination  of 
this  tissue  in  mammals  the  animal  from  which  the 
preparation  is  to  be  taken  should  have  been  killed 
some  hours  previously,  in  order  to  obviate  the 
shrinking  and  contraction  which  would  otherwise 
take  place  in  a  freshly-severed  portion  of  muscle 
placed  in  fluid,  and  would  obscure  many  of  the 
structural  appearances.  A  small  longitudinal  shred 
is  torn  or  stripped  oft'  from  any  muscle  of  the  limbs 
or  trunk,  placed  in  a  drop  of  serum  upon  a  slide, 
and  the  fibres  are  slowly  and  carefully  separated 
from  one  another,  one  by  one,  for  as  great  a  length 
as  possible.  A  very  small  slip  of  blotting-paper  is 
then  placed  by  the  side  of  the  preparation  to  avert 
the  pressure  of  the  cover-glass  (a  hair  is  not,  in  this 


VOLUNTARY    MUSCLE.  115 

instance,  thick  enough  for  the  purpose),  and  the 
latter  is  then  laid  on,  and  the  preparation  examined 
first  with  a  low  power,  to  make  out  the  shape  and 
extent  of  the  fibres,  and  afterwards  very  carefully 
with  an  ordinary  high  power,  and  eventually  with 
as  high  a  power  as  it  is  possible  to  obtain.  In  this 
preparation  almost  all  that  is  known  of  the  struc- 
ture of  mammalian  muscle  may  be  made  out — the 
delicate  sarcolemma  with  the  muscle  nuclei  imme- 
diately beneath  it,  which  look  clear  and  oval  when 
the  upper  surface  of  a  fibre  is  exactly  focussed,  fusi- 
form when  seen  at  the  edge,  the  dark  cross-stripes 
of  the  muscular  substance,  seen  by  careful  observa- 
tion with  a  very  high  power  to  be  pervaded  by 
parallel  rod-shaped  particles,  the  clear  stripes  bi- 
sected by  an  intermediate  dotted  line,  and  finally  a 
longitudinal  striation  throughout  the  fibre,  which 
is  the  better  seen  in  proportion  as  the  •  transverse 
striping  is  less  marked. 

Preparation  4. — For  the  purpose  of  bringing  the 
muscle-nuclei  more  distinctly  into  view,  another 
piece  may  be  prepared  in  a  drop  of  dilate  acetic  acid 
(one  part  in  200  of  water).  The  acid  swells  up  and 
softens  the  interstitial  connective  tissue,  so  that  the 
fibres  are  more  readily  separated  the  one  from  the 
other,  and  it  is  found  that,  whereas  in  the  former 
preparation  the  muscle-nuclei  could  only  be  made 
out  by  exercising  the  greatest  care  and  attention, 
they  are  now  extremely  obvious,  studding  the  fibres 
at  intervals,  but  all  of  them  quite  at  the  surface  of 
the  muscle  under  the  sarcolemma. 

Preparation  5. — When  the  frog's  muscles  are 
prepared  in  like  manner  with  acetic  acid,  the  nuclei 
are,  on  the  contrary,  seen  to  be  embedded  here  and 
there  in  the  thickness  of  the  fibre  also. 

Preparation  6. — The  sarcolemma  is  extremely 
delicate  in  mammalian  muscle,  and  although  it  may 
with  care  be  made  out  even  in  the  fresh  preparation, 
is  nevertheless  much  more  easily  demonstrated  in 
the  muscles  of  the  lower  vertebrata,  the  fro^,  for 


116  PRACTICAL    HISTOLOGY. 

example.  With  this  end  a  piece  of  the  muscle  is 
teased  in  a  drop  of  salt  solution.  This  fluid  has  the 
effect  of  gradually  disintegrating  the  proper  sub- 
stance of  the  muscle,  so  that  the  latter  tends  to 
break  down  into  a  clear  fluid  with  numerous  fine 
granules  suspended  in  it,  and  exhibiting  an  active 
Brownian  movement.  This  process  of  disintegration 
generally  begins  at  places  where  the  fibres  have  been 
touched  by  the  needles  in  the  process  of  separation, 
and  if  the  muscle  be  fresh,  the  contractile  substance 
breaks  and  shrinks  away  at  these  places,  leaving  the 
clear  sarcolemma  bridging  across  the  interval. 

Preparations  7  and  8.  Disks  and  Fibrils.— In 
order  to  exhibit  the  manner  in  which  muscular  tissue 
tends  to  break  up  into  either  disks  or  fibrils  accord- 
ing to  the  nature  of  the  reagent  to  the  action  of 
which  it  is  submitted,  two  pieces  of  muscle  are  taken 
for  an  animal  that  has  been  dead  some  hours,  and 
are  placed  for  a  week,  the  one  in  a  solution  of  hydro- 
chloric acid  (1  in  50),  the  other  in  a  solution  of 
chromic  acid  (1  in  200).  Small  portions  are  then 
broken  up  as  finely  as  possible  with  needles  upon 
separate  glass  slides.  The  fibres  from  the  hydro- 
chloric acid  are,  many  of  them,  found  to  cleave  into 
transverse  clear  disks,  some  of  which  will  be  noticed 
lying  flat,  others  seen  edgeways ;  whereas  in  those 
from  the  chromic  acid  there  is  no  tendency  what- 
ever to  form  such  disks,  but  on  the  contrary,  the 
muscular  fibres  tend  to  break  up  into  smaller  and 
smaller  longitudinal  fibrils. 

Preparation  9. — In  order  the  better  to  compare 
the  fibres  either  of  the  same  or  of  different  muscles 
as  regards  length  and  diameter,  and  to  see  their 
general  shape,  it  is  necessary  to  isolate  a  number  of 
them  in  their  whole  length.  For  this  purpose  the 
process  of  separation  by  the  aid  of  needles  is  some- 
what tedious,  and  we  must  turn  to  reagents  which 
will  dissolve  the  intermediate  connective  tissue 
which  binds  the  fibres  together,  whilst  maintaining 
them  intact.  Such  a  reagent  is  to  be  found  in  a 


LIVING    MUSCLE.  117 

solution  of  sulphurous  acid  (the  liquor  acidi  slil- 
phurosi  of  the  Pharmacopoeia  answers  very  well). 
The  muscle  is  placed  for  a  week  or  more  in  a  well- 
stoppered  hottle  containing  a  considerable  quantity 
of  the  acid,  and  is  kept  in  a  warm  chamber  (like 
that  shown  in  Fig.  19),  heated  to  about  #0°  or  40° 
Centigrade.  This  very  much  facilitates  the  process 
of  maceration,  so  that  after  the  time  stated  a  mere 
gentle  shaking  of  the  bottle  is  sufficient  to  cause  the 
muscle  to  break  up  in  great  measure  into  its  consti- 
tuent fibres.  Some  of  these  may  then  be  removed, 
placed  side  by  side  in  water  or  weak  glycerine  on  a 
slide,  and  covered,  with  the  usual  precautions  to 
obviate  the  pressure  of  the  cover-glass.  It  will  be 
found  that  the  muscular  substance  has  acquired,  in 
consequence  of  the  maceration,  a  granular  aspect, 
and  that  the  usual  structural  appearances  are  for  the 
most  part  indistinct.  But  this  mode  of  preparation 
may  be  advantageously  employed  for  the  purposes  of 
measurement  and  comparison  of  size  and  form  of 
fibres  from  different  regions  of  the  body. 

Preparation  10.  Examination  of  muscular 
tissue  in  the  living  condition.  Ultimate  struc- 
ture of  a  muscular  fibre. — The  delicacy  of  the 
structural  elements  of  the  striated  muscular  fibres 
of  the  vertebrata,  and  the  difficulty  of  maintaining 
the  fibres,  when  sufficiently  isolated,  in  the  living 
condition,  have  baffled  all  attempts  that  have  hitherto 
been  made  to  satisfactorily  determine  their  ultimate 
structure  before  it  has  become  altered  either  by  the 
death  of  the  tissue  or  the  action  of  reagents.  But 
the  muscular  tissue  of  some  of  the  in  vertebra!  a, 
particularly  of  insects  and  Crustacea,  bears  an  exact 
resemblance  to  the  transversely  striped  contractile 
tissue  of  the  higher  animals,  while  at  the  same  time 
the  appearances  are  better  marked,  and  separated 
portions  of  the  muscle  are  readily  prepared,  and  may 
be  examined  by  the  highest  powers  of  the  microscope 
whilst  still  living  and  freely  contractile. 

The  most  convenient  of  these  animals  to  employ 


118  PRACTICAL    HISTOLOGY. 

for  the  purpose  is  the  common  great  water-beetle 
(Dytiscus  marginalis),  and  the  muscles  of  the  limbs, 
especially  the  forelegs  of  the  males,1  which  are  strong 
and  well-developed,  should  be  chosen.  A  slide  and 
cover-glass  having  been  cleaned,  and  a  lance-pointed 
needle  or  a  scalpel  and  fine  scissors  and  forceps  being 
at  hand,  one  of  these  limbs  is  cut  off  at  the  joint 
nearest  the  body,  wiped  with  blotting-paper  to  free 
it  from  the  acrid,  strongly-smelling  secretion  which 
the  insect  emits,  and  the  chitinous  integument  of  the 
limb  is  then  slit  longitudinally  so  as  to  expose  the 
pale  soft-looking  muscle  within.  Then,  the  member 
being  firmly  held  on  to  the  table  with  forceps  or  the 
fingers,  as  much  as  possible  of  this  muscular  tissue 
is  gouged  out  with  the  lance-headed  needle  or  scalpel- 
point,  and  transferred  to  the  slide.  The  muscular 
fibres  are  quickly  separated  somewhat  from  one 
another  by  needles,  desiccation  being  prevented  by 
breathing  once  or  twice  upon  the  preparation,  and 
the  cover-glass  is  immediately  laid  on  without  the 
addition  of  any  fluid  other  than  that  which  naturally 
moistens  the  tissue.  The  preparation  is  to  be  ex- 
amined at  once  with  the  highest  available  power. 
It  is  difficult  to  make  out  the  details  of  the  structure 
with  a  combination  magnifying  less  than  1000  dia- 
meters, and  the  defining  power  must  be  of  the  best. 
If  the  object  has  been  quickly  enough  prepared, 
numerous  fibres  will  be  found  which  show  the  suc- 
cessive series  of  minute  muscle-rods,  corresponding 
in  position  with  the  darker  cross  stripes  of  the  mus- 
cle, and  prolonged  into  the  clear  stripes,  where  they 
end  with  knobbed  extremities,  the  juxtaposition  of 
Avhich  in  rows  side  by  side  gives  the  semblance  of  a 
dotted  line  in  each  clear  stripe,  single  or  double  accord- 
ing as  the  fibre  is  more  or  less  extended.  Other  fibres 

1  The  males  are  readily  distinguished  from  the  femnles  by  their 
smooth  wing-covers,  whereas  those  of  the  females  are  rigid  and 
furrowed  longitudinally,  as  well  as  by  their  short  strong  fore- 
limbs  with  sucker-like  extremities. 


LIVING    MUSCLE.  119 

may  be  seen  in  which  the  dotted  line  is  absent,  the 
muscle  rods  not  being  enlarged  at  their  extremities; 
and  these  being  placed  end  to  end  in  the  successive 
series  give  an  appearance  of  long,  jointed  fibrils  ex- 
tending in  the  direction  of  the  length  of  the  fibre. 
In  such  fibres  the  cross-striped  appearance  is  almost 
entirely  lost,  for  there  are  no  intervening  clear  striae, 
the  appearance  of  which  in  the  other  fibres  is  proba- 
bly due  to  the  presence  of  the  rows  of  strongly  refract- 
ing dots. 

That  the  bright  appearance  of  the  clear  striae  may  be 
produced  by  and  dependent  on  the  rows  of  minute  globu- 
lar ends  of  the  muscle  rods  is  shown  by  the  following  ex- 
periment :  A  drop  of  water  is  placed  on  a  slide,  and  a  very 
small  drop  of  oil  is  transferred  to  it  by  a  needle-point, 
and  is  then  thoroughly  whipped  up  with  it,  so  as  to  reduce 
the  oil  to  as  minute  globules  as  possible.  The  prepara- 
tion is  then  covered  and  examined  with  the  same  magni- 
fying power  as  was  used  for  the  water-beetle  muscle.  The 
smallest  of  the  oil-globules,  like  the  dots  in  the  muscle, 
look  like  mere  dark  specks,  and  it  will  be  noticed  that 
each  is  surrounded  by  a  bright  area,  an  effect  always  pro- 
duced by  highly  refracting  bodies  when  examined  in  a 
less  refracting  medium.  And  if  we  imagine  a  number  of 
such  little  oil-specks  to  be  placed  in  a  row,  there  would 
be  a  bright  band  on  either  side  of  the  rows  of  dots  in  the 
water-beetle's  muscle. 

Contraction  of  muscle  — In  the  perfectly  fresh  pre- 
parations of  the  insect's  muscular  tissue,  spontaneous 
waves  of  contraction  may  be  seen  passing  from  end  to  end 
of  many  of  the  muscular  fibres,  and  a  general  idea  of  the 
phenomena  which  accompany  the  contraction,  such  as 
thickening  of  the  part  of  the  fibre  at  the  time  the  wave  is 
passing,  and  approximation  of  the  cross-striae,  may  be 
obtained.  But  the  contraction  proceeds  too  quickly  for 
the  details  of  the  process  to  be  watched,  and  the  fibres 
are  not  sufficiently  isolated;  moreover,  it  is  impossible  to 
s&y  of  the  particular  fibre  under  examination  whether  a 
contractile  wave  is  about  to  pass  along  it  or  not. 

Preparation  11. — In  order  to  get  the  fibres  more 
under  command,  the  preparation  must  be  made  rather 
differently.  For  the  purpose  of  inducing  the  muscular 


120  PRACTICAL    HISTOLOGY, 

tissue  under  examination  to  contract  at  any  given  moment, 
a  gas-chamber  (see  Pig.  11)  is  made  use  of,  and  a  trace  of 
the  vapor  of  alcohol  is  made  to  pass  over  the  preparation, 
at  the  moment  when  it  is  wished  to  observe  the  contrac- 
tion of  a  particular  fibre.  The  mode  of  obtaining  and 
preparing  the  tissue  is  the  same  as  before,  except  that  the 
piece  scooped  out  of  the  freshly-excised  lirnh  is  placed 
upon  a  cover-glass  instead  of  on  a  slide,  and  the  fibres  are 
rather  more  freely  separated.  The  cover-glass  is  then  in- 
verted over  the  putty  ring  of  the  gas-chamber,  at  the  bot- 
tom of  which  a  drop  of  water  has  as  usual  been  previously 
placed.  The  preparation  is  searched  for  a  portion  of  a 
muscular  fibre  which  happens  to  be  conveniently  isolated, 
and  which  shows  well  the  normal  structure  of  the  living 
tissue.  This  having  been  brought  under  observation  with 
the  highest  magnifying  power,  a  little  air  charged  with 
the  vapor  of  rectified  spirit  which  has  been  previously 
poured  into  the  vapor-bottle  is  blown  into  the  gas-cham- 
ber; as  soon  as  the  contraction  which  results  is  over,  the 
vapor  must  be  replaced  by  air  again,  so  that  the  vitality 
of  the  tissue  is  not  too  soon  destroyed.  If  the  prepara- 
tion is  very  fresh,  the  contraction  is  generally  so  sudden 
that  it  is  impossible  to  follow  the  details  of  the  process ; 
but  after  a  time  the  tissue  responds  less  actively  to  the 
stimulus,  and  then  with  care  it  is  possible  to  make  out 
the  changes  which  are  happening  in  the  form  of  the  mus- 
cle rods,  and  in  the  consequent  relative  arrangement  of 
the  cross  stripes  of  the  fibre.  Numerous  trials,  if  neces- 
sary on  fresh  preparations,  may  have  to  be  made  before 
a  successful  result  is  arrived  at ;  for  various  circumstances, 
especially  the  shifting  of  the  fibres  during  their  contrac- 
tion, may  tend  to  vitiate  the  observation. 

Preparation  12.    Examination  of  muscular 

tissue  by  polarized  light. — The  polarizing  micro- 
scope is  nothing  else  than  the  ordinary  microscope  with 
the  addition  of  two  Nicol's  prisms,  one  placed  below  the 
object  and  another  above  the  ocular ;  the  upper  one  is 
generally  mounted  in  combination  with  a  low  ocular,  so 
that  it  is  not  necessary  to  use  the  ordinary  eye-piece. 
The  light,  coming  from  the  mirror,  becomes  polarized  as 
it  passes  through  the  lower  Nicol  (the  polarizer).  If  now 
the  upper  Nicol  (the  analyzer)  be  slowly  turned  round 
as  it  is  being  looked  through,  it  will  be  found  that  there 


POLARIZING    MICROSCOPE.  121 

are  two  positions  in  which  the  field  is  quite  dark  ;  that 
is  to  say,  the  polarized  ra}Ts  are  entirely  cut  off.  By  ob- 
serving now  the  relations  of  the  prisms  at  these  positions 
of  total  darkness,  it  will  be  found  that  their  planes  of 
polarization — as  shown  by  the  way  in  which  the  prisms 
are  cut — are  at  right  angles  to  one  another.  In  all  inter- 
mediate positions  a  greater  or  less  amount  of  light  is 
enabled  to  traverse  the  analyzer.  But  if  any  object 
which  possesses  the  property  of  refracting  light  doubly 
is  placed  upon  the  stage  of  the  microscope,  and  examined, 
and  if  then  the  field  is  made  dark  by  turning  the  ana- 
lyzer, it  will  lie  found  that  the  doubly  refracting  substance 
remains  bright,  unless  it  happen  so  to  lie  that  its  optic 
axis  is  parallel  with  the  plane  of  polarization  of  either 
Nicol.  And  if  the  object  be  a  muscular  fibre  at  rest,  the 
whole  fibre  will  appear  bright  and  doubly  refracting, 
whereas  if  it  be  in  the  state  of  contraction  the  bright 
stripes  only  will  allow  the  light  to  pass,  the  dark  stripes 
in  this  condition  of  the  fibre  being  singly  refracting. 

These  observations  are  best  made  upon  the  living 
muscle  of  the  water-beetle  by  aid  of  the  gas-chamber. 
The  portion  of  fibre  under  observation  should  be  quite 
free  and  not  overlaid  by  other  fibres.  The  change  in  the 
optical  condition  of  the  fibres  which  ensues  on  contrac- 
tion ma}',  if  due  care  and  patience  be  exercised,  in  this 
way  be  made  out.  The  results  arrived  at  by  the  exami- 
nation of  portions  of  the  tissue  which  have  been  hardened 
in  alcohol,  and  mounted  in  glycerine  or  Canada  balsam, 
although  more  easily  seen,  are  less  trustworthy,  since 
the  muscular  fibres  are  liable  to  undergo  considerable 
changes  after  death,  and  under  the  action  of  re-agents. 

A  pretty  modification  may  be  made  by  substituting  a 
thin  piece  of  mica  for  the  covering  glass.  This  causes 
the  field  of  view  to  become  tinted,  the  particular  color 
varying  with  the  thickness  of  the  mica,  and  the  relative 
position  of  its  optic  axis  to  those  of  the  Nicols,  and  any 
doubly  refracting  substance  which  is  now  examined 
assumes  the  color  which  is  complementary  to  that  of  the 
field.  The  object  of  the  revolving  stages  with  which  the 
larger  microscopes  are  generally  fitted  is  to  enable  the 
observer  to  modify  the  position  of  the  optic  axis  of  the 
tissue  which  is  being  examined,  with  relation  to  those  of 
the  Nicols  ;  and  it  also  serves  when  the  mica  is  used  to 
11 


122  PRACTICAL    HISTOLOGY. 

change  in  like  manner  the  relative  position  of  the  optic 
axis  of  this  also,  and  thus  to  modify  the  color  of  the 
field  of  view. 

Transversely  striated  muscle  is  not  by  any  means  the 
only  tissue  which  is  doubly  refracting,  for  the  property 
is  possessed  by  the  white  fibrils  of  connective  tissue,  and 
by  bone,  as  well  as  by  the  plain  muscular  fibre  cells. 
But  it  is  the  only  one  which  under  certain  conditions 
exhibits  alternate  bands  of  singly  and  of  doubly  refract- 
ing substance.  It  lias,  however,  been  pointed  out  by 
Ranvier  that  it  is  rather  the  conditions  of  growth  and 
formation  of  a  tissue  than  differences  of  structure  which 
tend  to  determine  differences  in  the  optical  properties  of 
the  substance  of  which  it  may  be  composed  And  he 
instances  the  case  of  cartilage,  the  matrix  of  which, 
although  undoubtedly  composed  of  the  same  substance 
throughout,  is  doubly  refracting  in  those  parts  where  the 
cells,  either  from  pressure  or  in  progress  of  growth,  have 
come  to  assume  either  a  flattened  or  elongated  shape, 
singly  refracting  where  they  remain  rounded. 

Preparation  13.  Transverse  section  of  mus- 
cle.— In  examining  the  fresh  muscle  of  the  water- 
beetle,  in  either  of  the  ways  above  described,  it  may 
happen  that  a  fibre  is  seen  which  is  turned  up 
towards  the  observer,  and  in  which,  therefore,  by 
focussing  the  vertical  part  with  the  fine  adjustment, 
a  view  may  be  obtained  just  as  if  the  fibre  had  been 
cut  transversely.  It  is  then  said  to  be  seen  in  optical 
section  ;  and  for  muscular  tissue  this  is  the  only  way 
in  which  a  section  of  the  fibres,  whilst  they  are  quite 
unaltered,  can  be  observed.  Nevertheless,  when  it 
is  not  so  much  the  structure  of  the  fibres  but  their 
shape  and  mode  of  arrangement  into  bundles  that  is 
to  be  the  subject  of  investigation,  the  tissue  may  be 
hardened  by  weak  chromic  acid  (}  per  cent.),  or  by 
alcohol,  and  thin  transverse  sections  may  be  cut 
with  a  razor  and  mounted  in  glycerine,  with  or 
without  previously  staining  in  logwood.  There  is 
another  method,  that  of  freezing^  which  has  luvn 
much  recommended  for  hardening  fresh  muscular 


SECTION    OF    FROZEN    MUSCLE.  123 

tissue  in  order  to  obtain  transverse  sections  of  the 
fibres  in  wbat  has  been  thought  a  completely  un- 
altered state.  It  may  be  doubted,  though,  whether 
the  muscular  substance  does  not  undergo  some 
structural  alteration  either  in  the  freezing  or  in  the 
subsequent  thawing.  Yet,  as  it  is  frequently  em- 
ployed for  this  tissue,  and  is  of  considerable  value 
in  special  investigations  into  the  structure  of  other 
tissues  and  organs,  the  method  may  be  here  de- 
scribed. 

Preparation  14.  Section  of  frozen  muscle. 
— The  best  and  simplest  mode  of  freezing  a  tissue 
is  that  recommended  by  Urban  Pritchard.  A  solid 
cylinder  of  copper  (1£  inch  wide  and  3  inches  long), 
with  flat  ends,  is  placed  for  a  few  minutes  in  a 
freezing  mixture  composed  of  equal  parts  of  pounded 
ice  and  salt,  by  which  it  is  cooled  to  considerably 
below  the  freezing-point.  The  copper  cylinder  is 
then  removed,  wiped  quite  dry  with  a  cloth,  and  a 
piece  of  flannel  is  wrapped  three  or  four  times  round 
it,  leaving  one  of  the  flat  ends  exposed.  The  muscle, 
which  may  conveniently  be  taken  from  the  leg  of  a 
frog,  is  cut  across  near  its  middle  and  one  of  the 
halves  is  placed  with  its  cut  surface  on  the  flat  end 
of  the  copper  block  in  a  drop  of  gum  arabic  solution. 
This  as  well  as  the  tissue  very  speedily  becomes 
frozen  throughout,  adheres  to  the  copper,  and  then, 
the  block  being  held  in  the  left  hand,  sections  are 
made  of  the  muscle  with  a  dry  razor,  which  should 
have  been  previously  cooled  by  contact  with  the 
vessel  containing  the  freezing  mixture,  and  the 
sections  are  at  once  transferred  to  a  slide  and 
covered,  either  in  serum,  or,  better,  without  the 
addition  of  any  fluid. 

Sections  made  by  freezing  possess  the  advantage 
that,  since  they  have  not  been  submitted  to  any 
coagulating  reagent,  they  may  be  treated  with 
nitrate  of  silver,  chloride  of  gold,  and  other  fluids 
which  are  of  value  only  when  the  tissues  are  per- 
fectly fresh. 


124  PRACTICAL    HISTOLOGY. 

Preparations  15  and  16.  Termination  of 
muscle  in  tendon. — It'  the  tendons  of  a  mouse's 
tail  are  forcibly  drawn  out,  after  nipping  off  the  end 
of  the  tail  in  the  manner  described  in  the  account 
of  the  preparation  of  tendon  (p.  83),  it  will  generally 
be  found  that  portions  of  a  number  of  small  mus- 
cular fibres  are  adherent  to  each  tendon,  for  the 
fibres  have  their  insertions  into  the  tendon,  and  are 
ruptured  by  the  force  employed.  These  ends, 
mounted  in  serum,  serve  conveniently  for  the  study 
of  the  mode  in  which  the  fibres  of  a  muscle  termi- 
nate in  a  tendon,  when  the  fibres  of  the  latter  run 
in  the  same  direction  of  those  of  the  muscle.  But, 
easy  as  the  tissue  is  to  prepare,  the  observation  is 
complicated  by  the  fact  that  the  muscular  fibres 
form  generally  somewhat  of  a  clump  as  they  pass  to 
end  in  the  tendon.  The  preparation  may  be  im- 
proved by  being  stained  with  picrocarmine  solution. 
This  colors  muscular  tissue  yellow,  tendinous  tissue 
red,  so  that  the  distinction  between  the  two  is  made 
more  obvious.  It  is  best  to  take  a  few  freshly 
drawn-out  tendons,  and  to  mount  their  ends  in  a 
drop  of  the  picrocarmine  solution,  surrounding  the 
edges  of  the  cover-glass  with  melted  paraffin  to  pre- 
vent evaporation  of  the  liquid. 

It  is  easier  to  make  out  the  way  in  which  the 
fibres  of  a  very  thin  fiat  muscle,  such  as  the  subcu- 
taneous muscle  over  the  frog's  sternum,  terminate 
in  the  fibrous  tissue  to  which  the  muscle  is  attached. 
Here  the  fibres  of  the  tendon  do  not  take  the  same 
direction  as  the  muscular  fibres.  A  frog,  having 
been  killed  by  destroying  the  brain  and  spinal  cord, 
is  laid  upon  its  back,  and  the  skin  over  the  throat 
is  reflected  downwards  towards  the  sternum.  The 
flat  muscle  w^hich  is  thereby  brought  to  view  passing 
to  the  skin  is  snipped  across  the  middle  with  scissors, 
seized  on  the  cutaneous  side  of  the  snip  with  forceps, 
and  a  square  piece,  including  its  insersion  into  the 
skin,  is  cut  out.  It  is  placed  as  flat  as  possible  upon 
a  dry  slide,  the  curled-up-  edges  being  turned  down 


TERMINATION    OF    MUSCLE    IN    TENDON.      125 

by  the  aid  of  mounted  needles,  and  a  drop  of  serum 
having  been  placed  upon  a  cover-glass,  this  is  in- 
verted over  the  preparation.  The  rounded  ends  of 
the  muscular  fibres  are  seen  fitting  into  little  depres- 
sions in  the  fibrous  tissue,  and  with  prolongations 
from  this  passing  around  and  between  them. 

The  bloodvessels  of  muscle  will  be  studied  later,  after 
the  methods  of  injecting  and  of  cutting  sections  have  been 
described.  The  mode  of  termination  of  nerves  in  volun- 
tary muscle  will  also  be  most  advantageously  deferred 
until  the  preparation  of  the  nerves  themselves  has  been 
treated  of. 


126  PRACTICAL    HISTOLOGY. 


CHAPTER  VII. 

NERVOUS  TISSUE. 

Preparation  1.  Medullated  nerve-fibres. — For 

the  study  of  the  medullated  nerve-fibres  a  piece  of 
one  of  the  ordinary  nerves — those  of  the  limbs,  for 
example — may  be  cut  out  from  any  recently-killed 
animal.  If  the  nerve  be  a  large  one,  a  thin  strip  only 
should  be  used,  preferably  taken  from  the  interior 
after  the  piece  has  been  torn  longitudinally  into  two 
halves  by  fine  forceps.  The  strip  is  to  be  placed  on 
a  slide  in  a  little  serum  or  salt  solution,  and  care- 
fully separated  as  finely  as  possible.  This  separation 
must  be  effected,  not  by  seizing  the  piece  anywhere 
and  tearing  it  up  at  random,  but  by  inserting  tine 
needles  into  it  near  one  end  and  gently  drawing  them 
asunder,  so  that  the  piece  is  split  into  two.  Repeating 
this  process  a  number  of  times  on  the  resulting  pieces 
the  nerve  will  be  eventually  separated  into  very  tine 
bundles  of  fibres,  together  with  a  number  of  more  or 
less  isolated  fibres,  which  are  still  nearly  straight 
and  uninjured,  except  near  one  end.  The  prepara- 
tion may  then  be  covered,  and  the  general  character 
and  appearance  of  the  fibres  investigated.  To  see 
the  nodes  of  Ranvier  well,  a  tolerably  large  fibre, 
free  for  a  considerable  part  of  its  length,  should  be 
chosen,  and  by  moving  the  slide  it  should  be  care- 
fully followed  with  an  ordinary  high  power.  It 
will  be  found  that  at  definite  and  not  very  close  in- 
tervals along  the  fibre  the  double-contoured  medul- 
lary sheath  fails  altogether,  and  the  axis-cylinder 
alone  continues  the  nerve  at  these  points.  It  is  not 
very  easy  to  see  the  oval  nucleus  in  the  middle  of 
each  segment  in  the  fresh,  unstained  preparation. 


SHEATH    OF    NERVE.  127 

Preparation  2, — Osmic  acid  possesses  the  pro- 
perty of  staining  fatty  matters  of  an  inky  black 
color.  Since  the  medullary  sheath  of  the  nerves  is 
mainly  composed  of  a  phosphuretted  fat,  this  becomes 
accordingly  stained  by  the  acid,  whilst  the  other 
parts  are  left  of  a  grayish  tinge.  Moreover,  the 
regular  breaks  in  the  fatty  sheath — the  nodes  of 
Ranvier — are  by  this  means  brought  into  promi- 
nence, owing  to  the  complete  intermission  of  the 
darkly  stained  medullary  substances  at  those  places. 
At  the  same  time  the  axis-cylinder  can  be  distinctly 
made  out  crossing  the  interval,  and  the  primitive 
sheath,  or  sheath  of  Schwann,  can  be  often  seen  both 
here  and  also  near  the  centre  of  each  of  the  segments 
into  which  the  fibre  is  divided,  where  it  is  bulged 
out  by  the  presence  of  the  oval  nucleus.  The  method 
for  preparing  these  osrnic  preparations  is  as  follows : 
From  an  animal  which  has  been  quite  recently  killed, 
a  small  nerve,  not  larger  in  diameter  than  an  ordi- 
nary thread,  is  chosen,  and  a  piece  about  half  an  inch 
long  is  cut  out,  but  in  doing  so  care  must  be  taken 
not  to  drag  upon  or  injure  the  nerve  more  than  is 
absolutely  necessary.  The  piece  is  placed  for  four 
hours  in  a  little  covered  glass  pot  contaiug  a  drop 
or  two  of  a  1  per  cent,  solution  of  osmic  acid  ;  it  is 
then  transferred  to  water  for  an  hour,  and  finally 
placed  in  a  mixture  of  glycerine  and  water  (equal 
parts).  It  can  either  be  teased  in  this  at  once  or 
left  for  a  day  or  two  or  even  longer ;  in  preparing 
it,  the  same  precautions  must  be  used  as  were  recom- 
mended for  the  preparation  of  the  fresh  nerve  (see 
preceding  paragraph). 

Preparation  3.  Fibres  of  Remak.— To  see  the 
gray  or  non-medullated  fibres,  pieces  of  the  sympa- 
thetic nerve  are  taken  from  the  neck  of  an  animal 
and  prepared  and  examined  both  fresh  and  after 
treatment  with  osmic  acid,  in  the  same  manner  as 
the  cerebro-spinal  nerves. 

Mode  of  union  of  the  nerve-fibres  to  form 
the  nervous  cords. — In  the  several  teased  prepara- 


128  *       PRACTICAL    HISTOLOGY. 

tions  both  of  the  cerebro-spinal  and  of  the  sympa- 
thetic nerves  there  will  be  seen,  especially  in  those 
prepared  with  osmic  acid,  besides  the  actual  nerve- 
fibres,  in  the  first  place,  a  quantity  of  connective 
tissue,  for  the  most  part  of  the  nature  of  areolar 
tissue,  which  formed  a  general  ensheathment  for 
the  nerve,  and  sent  partitions  in  between  its  several 
bundles  or  funiculi ;  secondly,  the  special  sheaths  of 
the  funiculi,  which  become  torn  and  stripped  away 
in  the  process  of  teasing,  and  which  look  like  flat 
bands  composed  of  an  almost  homogeneous  substance, 
but  pervaded  by  a  network  of  fine  elastic  fibres  and 
with  round  or  ovalish  nuclei  scattered  upon  them 
here  and  there.  Thirdly,  there  will  be  seen  running 
along  close  to  and  surrounding  the  nerve-fibres  them- 
selves very  delicate,  nearly  straight  fibrils  of  con- 
nective tissue,  with  here  and  there  the  nucleus  of 
a  connective  tissue  corpuscle.  These  three  forms 
of  connective  tissue  represent  respectively  the  epi- 
rieurium  (cellular  sheath);  the  perineuriurn  (neuri- 
lemma);  and  the  endoneurium,  or  tissue  within 
the  funicular ;  but  their  relative  situation  and 
arrangement,  as  well  as  the  lamellated  structure  of 
the  perineurium,  can  only  be  properly  displayed  by 
transverse  sections  of  a  nervous  trunk,  the  mode  of 
preparing  which  will  not  be  treated  of  until  the 
general  directions  for  the  preparation  of  sections  have 
been  given. 

Preparation  4. — But  the  cell-outlines  on  the 
lamellae  of  the  perineurium  may  be  shown  by  the 
silver  method.  For  this  purpose  either  a  very  small 
nerve  is  chosen  and  a  piece  of  it  is  removed  and 
immersed  for  ten  minutes  in  half  per  cent,  silver 
nitrate  solution,  after  which  it  is  washed  in  distilled 
water  and  exposed  in  glycerine  to  the  light ;  or, 
should  it  be  wished  to  prepare  a  larger  nerve  con- 
sisting of  more  than  one  funiculus,  this  is  carefully 
dissociated  in  a  few  drops  of  the  silver  solution,  and 
after  a  like  treatment  is  also  to  be  mounted  in 
glycerine  and  exposed  to  the  light.  After  a  few 


NERVE -CELLS.  129 

minutes'  exposure  to  sunlight  the  preparations  may 
be  examined.  It  will  be  found  that  the  sheath  of 
each  funiculus  or  nervous  bundle  is  covered  by  large 
epithelioid  marking,  and  if  the  preparation  be  very 
successful,  two,  three,  or  even  more  layers  deep  of 
such  markings  may  be  counted  by  examining  with 
a  high  power  and  carefully  adjusting  the  microscope. 

In  addition  to  this  it  will  generally  be  found  that 
the  medullated  nerve-fibres  themselves  are  marked 
transversely  at  each  of  the  nodes  of  Ranvier  by  a 
dark  line,  or  rather  ring,  surrounding  the  axis- 
cylinder.  These  markings  appear  to  be  owing  to  the 
existence  of  a  substance  here,  between  the  segments 
of  the  nerves,  which,  like  the  intercellular  substance 
elsewhere,  has  an  affinity  for  the  metal.  Finally,  at 
many  of  the  nodes  of  feanvier,  particularly  if  the 
immersion  in  the  silver  solution  had  been  rather 
prolonged,  the  latter  will  have  penetrated  to  and 
become  reduced  in  the  substance  of  the  axis  cylinder, 
which  therefore  will  here  have  the  appearance  of  a 
dark  line  or  band  piercing  the  ring  of  interseg- 
mental  substance,  the  twTo  together  having  the  sem- 
blance, under  a  moderate  power,  of  a  little  black 
cross  upon  the  nerve  at  those  points.  Sometimes 
in  such  preparations  the  axis-cylinder,  where  stained 
at  the  nodes  by  the  reduced  silver,  presents  a  cross- 
striated  appearance,  but  the  meaning  of  this  is  not 
known. 

Nerve-  and  ganglion- cells  ;  structure  of  the 
nerve-centres. — To  study  the  exact  form  and  ap- 
pearance of  the  tissue  elements  of  the  nervous 
centres  they  must  be  as  much  as  possible  isolated 
and  examined  in  teased  preparations,  although  their 
position  and  local  relations  are  best  made  out  in 
sections  of  the  several  organs  in  which  they  occur. 
For  the  present  we  will  confine  ottrselves  to  a  descrip- 
tion of  the  best  methods  for  isolating  the  nerve-cells. 
For  this  purpose  the  tissue  is  treated  similarly  from 
whatever  part  of  the  brain  or  spinal  cord  the  piece 
to  be  examined  is  removed ;  but,  as  a  typical  example, 


130  PRACTICAL    HISTOLOGY. 

a  piece  of  the  spinal  cord  from  the  lumbar  region 
may  be  taken,  for  the  nerve-cells  are  here  very 
numerous  and  consequently  more  readily  found 
than  in  other  regions. 

Preparation  5, — The  human  spinal  cord  is,  if 
obtainable,  best  adapted  for  the  study  of  the  cells, 
for  they  are  readily  picked  out  under  the  dissecting 
microscope,  in  consequence  of  the  little  mass  of  dark 
pigment  which  each  contains.  If  a  piece  of  the 
human  spinal  cord  cannot  be  got,  the  spinal  marrow 
of  the  ox  or  sheep,  or  other  tolerably  large  animal, 
may  be  employed.  The  piece  to  be  macerated 
should  be  about  half  an  inch  long,  and  is  placed  for 
forty-eight  hours  in  weak  bichromate  of  potash 
solution  (1  in  800).  At  the  expiration  of  this  time 
it  is  taken  out  of  the  fluid,  and  with  the  point  of  a 
scalpel  or  of  fine  scissors  a  small  piece  of  the  gray 
matter  is  dug  out  from  the  anterior  cornu,  placed  in 
a  drop  of  water  upon  a  slide,  and  separated  with 
needles  into  three  or  four  pieces.  These  are  then 
placed  under  the  dissecting  microscope  and  carefully 
broken  up  further ;  those  parts  where  the  cells  are 
most  numerous  being  singled  out,  and  the  prepara- 
tion being  examined  every  now  and  then  on  the 
ordinary  microscope  with  a  low  power,  in  order  that 
the  progress  of  the  isolation  may  be  watched.  When 
as  much  as  possible  has  been  effected  in  the  way  of 
separating  the  nerve-cells  a  piece  of  hair  is  placed 
in  the  fluid  and  the  cover-glass  gently  superposed. 
The  cells  may  then  be  sought  with  a  low  power  and 
carefully  examined  with  a  higher  one. 

If  the  preparation  is  very  successful,  and  it  be 
desired  to  preserve  it,  keeping  the  cells  at  the  same 
time  as  much  as  possible  of  their  natural  aspect,  the 
best  plan  to  adopt  is  to  allow  a  drop  or  two  of  a  1 
per  cent,  solution  of  osrnic  acid  to  flow  in  at  the  edge 
of  the  cover-glass,  and  after  it  has  acted  upon  the 
tissue  for  an  hour,  by  which  time  the  cells  will  have 
'become  stained  of  a  dark  grayish  tint,  to  carefully 
run  through  from  the  same  side  first  a  little  distilled 


GANGLION    CELLS.  131 

water  to  wash  away  what  remains  of  the  acid,  and 
then  glycerine  to  preserve  the  preparation,  after 
winch  the  edges  of  the  cover-glass  may  be  cemented. 
Besides  the  nerve-cells,  which  in  a  carefully-prepared 
specimen  may  be  very  well  seen,  with  their  long 
branching  processes  extending  in  some  cases  far  be- 
yond the  field  of  the  high  power  objective,  some 
points  in  the  structure  of  the  nerve-fibres  of  the 
spinal  cord  can  be  well  made  out  in  these  prepara- 
tions. For  example,  it  may  be  readily  seen  that  the 
medullary  sheath  has  broken  away  in  many  parts 
from  the  white  nerve  fibres,  and  where  not  actually 
removed  has  become  swollen  and  coagulated  in 
irregular  masses  around  the  axis  cylinder,  changes 
which  could  hardly  have  taken  place  were  there  any 
structure  surrounding  the  nerve  fibres  such  as  the 
primitive  sheath  of  the  peripheral  nerves.  Further, 
where  the  axis-cylinders  are  in  this  manner  laid  bare, 
as  they  often  are  for  a  considerable  part  of  their 
length,  their  fibrillar  structure  can,  with  a  high 
power,  be  made  out  without  difficulty.  A  similar 
structure  can  also  he  seen  in  the  processes  of  the 
nerve  cells,  and  extending  from  them  through  the 
body  of  the  cell  itself. 

Preparation  6. — The  cells  from  the  ganglia, 
whether  spinal  or  sympathetic,  are  isolated  in  a 
manner  similar  to  that  employed  in  the  case  of  the 
spinal  cord,  except  that  the  period  of  maceration  in 
the  bichromate  solution  needs  to  be  longer,  owing 
to  the  much  larger  amount  of  connective  tissue  by 
which  the  nervous  elements  are  invested.  At  the 
same  time  it  is  quite  possible  to  get  a  certain  number 
of  the  cells  sufficiently  isolated,  even  from  the  fresh 
ganglion,  without  any  maceration;  for  each  cell 
being  loosely  contained  in  a  special  capsule  of  flat- 
tened cells,  it  readily  falls  out  when  the  nerve  fibres 
with  which  it  is  connected  are  ruptured.  But 
although  the  cells  themselves  of  the  ganglia  are 
readily  enough  separated,  it  is  very  difficult,  both 
in  the  fresh  and  in  the  macerated  preparations,  to 


132  PRACTICAL    HISTOLOGY. 

get  their  continuation  to  the  nerve  fibres,  these  being 
left  behind  for  the  most  part.  The  mode  of  perma- 
nently preserving  the  specimen  is  like  that  employed 
in  the  preceding  preparation,  except  that  the  cells 
may  with  advantage  be  colored  by  logwood  instead 
of  osrnic  acid. 

Terminations  of  the  nerves. — The  description 
of  the  mode  of  preparing  and  demonstrating  the 
terminations  of  nerve  fibres  in  various  special  parts 
of  the  body  will  be  deferred  until  those  parts  and 
organs  are  severally  treated  of,  but  the  Pacinian 
bodies,  in  which  many  of  the  sensory  fibres  end,  and 
the  end-plates  in  which  the  nerves  supplying  the 
voluntary  muscles  terminate,  may  be  now  prepared. 

Preparation  7. — The  Pacinian  bodies  are  very 
readily  found  in  the  cat's  mesentery.  Here  they  are 
at  once  seen  when  the  abdomen  is  opened  and  the 
membrane  is  held  up  against  the  light,  as  clear,  oval 
specks,  either  dotted  singly  here  and  there,  or  form- 
ing groups  of  two,  three,  or  more.  There  is  generally 
a  considerable  group  in  the  meso-rectum,  and  more- 
over they  are  here  usually  not  so  much  obscured  by 
the  adipose  tissue  as  in  the  mesentery  proper.  By 
far  the  best  general  idea  of  their  structure  and  the 
relation  they  have  to  the  nerve  fibre  entering  them 
is  obtained  from  their  study  in  the  fresh  condition, 
without  the  addition  of  reagents.  But  it  is  well  to 
separate  them  from  the  surrounding  tissue  of  the 
mesentery,  for  this  is  often  loaded  with  fat,  and, 
when  not,  the  fibrous  tissue  of  the  membrane  tends 
to  obscure  the  structure  of  the  little  bodies. 

In  order  to  isolate  one  of  them,  cut  out  the  piece 
of  the  mesentery  containing  it,  carrying  one  of  the 
cuts  close  along  the  edge  of  the  corpuscle.  Then 
place  the  excised  piece  upon  a  slide  in  a  drop  of 
serum,  and  without  actually  transfixing  the  Pacinian 
itself,  tear  away  the  investing  mesenteric  tissue  bit 
by  bit  under  the  dissecting  microscope.  It  will  be 
found  that  with  a  little  manipulation  the  corpuscle 
shells  out  as  a  lemon-shaped  body,  with  a  twisted 


THE    PACINIAN    CORPUSCLES.  133 

stalk  at  one  end.  It  is  to  be  examined  with  a  low 
power  to  make  sure  that  the  fat  is  entirely  removed  ; 
the  debris  may  then  be  wiped  away,  a  little  fresh 
serum  added,  a  narrow  slip  of  blotting-paper  placed 
close  to  the  corpuscle  to  avert  the  pressure  of  the 
cover-glass,  which  is  then  laid  on  and  the  specimen 
examined. 

In  these  fresh  preparations,  owing  to  the  extreme 
transparency  of  the  layers  of  the  capsule,  the  core  of 
the  corpuscle  with  the  central  fibre,  together  with 
the  mode  of  passage  of  the  nerve  fibre  into  this  at 
the  stalk,  can  be  made  out  better  than  after  the 
action  of  reagents.  And  still  more  so  if  the  outer 
layers  of  the  capsule  are  removed  altogether,  as  may 
readily  be  done  with  fine  needles  under  the  dissect- 
ing microscope,  leaving  only  the  core  and  the  closely- 
set  layers  of  the  capsule  which  immediately  surround 
it.  In  tearing  away  the  outer  layers  it  will  often 
happen  that  the  perineural  or  neurilemmal  sheath 
which  surrounds  the  nerve  fibre  as  this  passes  into 
the  corpuscle  is  torn  off  along  with  them,  since  they 
are  directly  continuous  with  it. 

These  fresh  preparations  of  the  Pacinians  are  very 
beautiful,  but  unfortunately  they  cannot  be  preserved 
in  that  state.  Treatment  with  glycerine  causes  the 
corpuscles  to  shrink  and  become  too  transparent, 
and  most  of  the  ordinary  staining  fluids  color  the 
core  too  deeply  and  obscure  the  termination  of  the 
nerve  fibre.  If  it  he  desired  to  preserve  any  such 
preparation  as  showing  some  one  or  more  points 
particularly  well,  the  cautious  employment  of  osmic 
acid  prior  to  mounting  in  glycerine  is  most  to  be 
recommended.  The  serum  in  which  the  corpuscle 
is  mounted  must  first  be  replaced  by  salt  solution; 
this  again  by  1  per  cent,  solution  of  osmic  acid,  and 
this  after  being  left  for  an  hour  or  more  in  contact 
with  the  preparation  by  distilled  water,  whilst  finally 
a  drop  of  glycerine  is  allowed  gradually  to  diffuse 
in  from  the  edge  of  the  cover-glass. 
12 


134  PRACTICAL    HISTOLOGY. 

Preparation  8. — The  structure  of  the  tunics 
which  form  the  lamellated  capsule  of  the  Pacinian 
body  is  not  well  shown  in  the  fresh  preparation, 
owing  to  the  transparency  of  the  object ;  indeed, 
it  is  the  lines  of  contact  between  successive  coats 
which  look  like  layers  of  the  capsule  ;  the  substance 
of  the  coat  being  clear  and  pellucid,  gives  the  notion 
of  an  intermediate  fluid.  In  order  to  show  their 
fibrous  connective  tissue  structure  (the  white  fibrils 
running  transversely,  and  collected  for  the  most 
part  near  the  surfaces  of  each  tunic,  and  the  elastic 
fibrils  forming  a  network  through  the  thickness) 
some  of  the  little  bodies  should  be  dissected  out  in 
the  way  above  described, -and  placed  for  ten  days  or 
a  fortnight  in  a  £  per  cent,  solution  of  chromic  acid. 
They  are  then  placed  on  a  slide  in  a  drop  of  water, 
and  with  fine  and  perfectly  clean  needles  are  broken 
up  under  the  dissecting  microscope  bit  by  bit,  com- 
mencing at  one  end  and  breaking  off  transverse 
pieces.  It  will  be  found  that,  owing  to  the  direction 
of  the  fibrils,  the  corpuscles  tend  to  break  across 
into  disk-like  portions.  The  core  does  not  share 
this  tendency,  but  this  is  of  little  consequence,  for 
it  is  not  well  displayed  in  these  preparations.  A 
piece  of  hair  having  been  added,  the  preparation  is 
covered  and  examined.  Small  fragments  will  pro- 
bably be  found  which  give  a  sectional  view  of  the 
tunics,  and  others  in  which  they  are  seen  fiat.  If 
a  little  logwood  solution  is  allowed  to  run  under 
the  cover-glass,  nuclei  on  the  surface  of  the  tunics 
may  here  and  there  be  stained. 

Preparation  9. — These  nuclei  belong  to  flattened 
epithelioid  cells,  which  cover  both  outer  and  inner 
surface  of  each  tunic,  and  which,  seen  in  profile,  are 
in  reality  the  well-defined  lines  seen  in  the  fresh 
Pacinian,  and  long  described  as  the  coats  themselves. 
The  outlines  of  these  cells  may  be  brought  into  view 
by  staining  with  nitrate  of  silver.  For  this  purpose, 
as  is  always  the  case  with  silver-preparations,  the 
tissue  must  be  fresh  and  unacted  on  previously  by 


MOTOR    END-PLATES.  135 

any  other  reagent.  One  or  two  corpuscles  are  to 
be  thoroughly  isolated  and  freed  from  surrounding 
mesenteric  tissue  and  fat ;  placed  for  ten  minutes 
in  silver  solution  (1  in  200);  washed  in  distilled 
water,  and  exposed  in  glycerine  to  the  sunlight 
until  of  a  grayish  color,  when  they  may  be  covered, 
and  examined. 

Preparation  10  — To  complete  the  study  of  the 
Pacinians,  sections  should  be  made  of  them,  but  as  they 
are  very  small,  it  will  be  best  to  defer  this  until  some  prac- 
tice has  been  attained  in  the  art  of  cutting  microscopic 
sections.  A  convenient  way  to  prepare  them,  in  order 
to  show  the  various  parts  to  advantage,  is  as  follows : 
A  very  small  piece  of  meso-rectum,  containing  several 
corpuscles  close  together,  is  cut  out  (if  such  can  be 
found  ;  if  not,  one  or  more  may  he  isolated  as  before), 
and  placed  in  a  small  beaker  containing  100  cub.  cent,  of 
a  weak  solution  of  acetic  acid  (1  in  200),  to  which  about 
5  c.  c.  of  the  ordinary  (^  per  cent.)  chloride  of  gold  solu- 
tion has  been  added.  The  tissue  is  kept  in  this,  in  the 
light,  for  three  or  four  days — until  it  has  become  of  a 
dark  violet  color;  it  is  then  placed  for  a  day  in  weak 
spirit,  and  then  in  strong,  and  two  or  three  days  later  is 
ready  for  embedding  and  cutting.  In  embedding  the 
piece  of  tissue,  it  should  be  so  placed  that  the  corpuscles, 
at  least  most  of  them,  are  cut  as  nearly  as  possible  trans- 
versely.1 

Preparation  11.— The  end-plates,  or  terminal 
expansions  of  the  motor  nerves,  are  difficult  to  find, 
and  so  soon  undergo  alteration  as  speedily  to  become 
unrecognizable.  On  this  account  it  is  necessary,  in 
searching  for  them,  to  employ  only  muscles  which 
are  absolutely  fresh.  In  mammalia  the  best  muscles 
to  choose  for  the  purpose  are  those  of  a  lamellar 
shape  and  with  short  fibres,  such,  for  instance,  as  the 
intercostals  of  small  animals.  The  muscular  fibres 

1  A  description  and  delineations  of  the  appearances  exhibited 
by  the  Pacinian  corpuscles  when  prepared  in  these  several  ways 
will  be  (bund  in  the  '•  Quarterly  Microscopical  Journal''  for  April, 

1875. 


136  PRACTICAL    HISTOLOGY. 

are  severed  close  to  their  attachments,  so  as  to  get 
them  in  their  whole  length,  and  the  small,  thin 
piece  of  muscular  tissue  obtained  is  quickly  trans- 
ferred to  a  slide,  and  mounted,  either  without  addi- 
tion of  fluid  or  in  a  small  drop  of  serum  which  is 
put  on  the  cover-glass  before  this  is  inverted  over 
the  preparation. 

This  should  now  be  thoroughly  searched  with  a 
good  immersion  objective  for  the  nerve  endings. 
Branches  of  the  intercostal  nerve  will  be  found 
running  across  the  direction  of  the  muscular  fibres. 
Starting  from  one  of  these,  trace  carefully  one  by 
one  the  single  nerves  which  pass  off  from  it.  It  will 
be  found  generally  that  they  branch  one  or  more 
times,  and  eventually  the  resulting  twigs  pass  off  to 
the  muscular  fibres,  each  fibre  receiving  one  of  the 
nerve-twigs.  They  retain  their  medullary  sheath 
until  the  muscular  fibre  to  which  they  are  attached 
is  reached,  when  the  sheath  suddenly  ceases  to  be 
visible,  and  it  is  by  following  the  single  fibres  until 
they  come  in  this  way  to  an  abrupt  termination, 
that  an  end-plate  may  be  met  with.  But  even  if  the 
place  where  the  nerve-fibre  joins  the  muscular  fibre  is 
arrived  at,  it  is  still  in  most  cases  difficult  to  make 
out  the  exact  mode  of  termination,  in  other  words, 
the  structure  of  the  plate.  The  utmost  that  can 
generally  be  seen  is  a  clump  of  clear,  round  nuclei 
embedded  in  a  granular  meterial.  The  difficulty 
arises  partly  from  the  readiness  with  which  these 
structures  undergo  alteration  after  removal  in  warm- 
blooded animals,  and  partly  from  the  fact  that  they 
are  often  obscured  by  super-  and  sub-jacent  muscular 
fibres  or  bloodvessels. 

Preparation  12. — In  the  common  lizard  (Lacerta 
agilis)  the  end-plates  may  be  much  more  easily 
found  and  satisfactorily  seen,  but  still  the  utmost 
care  must  be  taken  in  the  preparation.  The  animal 
having  been  decapitated  and  the  trunk  pinned  out 
upon  a  cork,  a  piece  of  one  of  the  limb-muscles — 
including  the  whole  length  of  the  fibres — is  removed 


MOTOR    END-PLATES. 

and  placed  on  a  slide  in  a  drop  of  serum.  The  fibres 
are  then  isolated  under  the  dissecting  microscope  as 
carefully  and  completely  as  may  be,  and  a  piece  of 
paper  or  a  hair  having  been  added  to  avert  pressure 
on  the  tissue,  it  is  covered  and  the  fibres  are  ex- 
amined with  an  immersion  along  their  whole  length. 
Of  course,  if  no  end-plates  can  be  found  in  the  first 
specimen  another  must  be  taken,  but  it  will  gene- 
rally not  be  very  long  before  one  is  found,  either  at 
the  edge  of  a  fibre,  and  therefore  seen  in  profile,  or 
on  the  surface,  and  seen  flat. 

Unfortunately,  a  method  has  not  yet  been  dis- 
covered by  which  they  may  be  well  preserved.  That 
which  on  the  whole  answers  the  best  is  to  treat  the 
preparation  with  1  per  cent,  osmic  acid,  having  first 
washed  away  the  serum  by  salt  solution.  After  tho 
osmic  has  been  about  an  hour  in  contact  with  the 
tissue,  it  may  be  washed  away  and  glycerine  sub- 
stituted. In  this  way  the  form  of  the  little  end- 
plate  or  prominence  is  well  preserved,  and  the 
sudden  termination  of  the  medullary  sheath  of  the 
nerve  fibre  well  shown,  since  this  becomes  blackened 
by  the  osmic  acid,  but  the  substance  of  the  end-plate 
becomes  dark  and  granular,  so  that  the  nuclei  are 
with  difficulty  seen. 


12* 


138  PRACTICAL    HISTOLOGY. 


CHAPTER    VIII. 

THE  BLOODVESSELS. 

Preparation  1.  The  larger  bloodvessels.— 
The  epithelioid  lining  can  only  be  properly  demon- 
strated in  fresh  bloodvessels  stained  by  nitrate  of 
silver.  For  this  purpose  a  piece  of  a  large  vessel- 
artery  or  vein — is  obtained,  either  from  a  recently - 
killed  animal  or  from  an  amputated  limb,  and  hav- 
ing been  slit  open  with  scissors  it  is  pinned  on  to  a 
cork  with  the  inner  surface  uppermost.  Care  must 
be  taken  in  doing  so  not  to  rub  this  surface  in  any 
way.  A  little  distilled  water  is  then  spirted  over 
the  preparation  from  a  wash-bottle,  with  the  object 
of  removing  any  blood  or  serum  that  may  remain 
on  the  wall  of  the  vessel,  and  a  few  drops  of  half 
per  cent,  nitrate  of  silver  solution  are  allowed  to 
flow  over  the  surface.  After  a  minute  it  is  again 
washed  with  distilled  water,  and  is  then  put  at  once 
into  a  beaker  of  spirit  and  placed  in  the  sunlight. 
After  a  time  the  surface  will  have  acquired  a  gray- 
ish tinge,  with  a  browner  patch  here  and  there ;  it 
may  now  be  removed  from  the  window,  but  should 
be  left  in  spirit  until  the  next  day,  when  it  will  be 
hard  enough  to  enable  thin  sections  to  be  made  with 
a  razor  from  the  inner  surface.  In  order  to  cut  them 
it  will  be  found  convenient  to  remove  the  piece  of 
bloodvessel  from  the  cork,  and  to  hold  it  by  one  end 
by  the  thumb  and  fingers  of  the  left  hand,  so  that 
the  piece  rests  by  its  outer  surface  on  the  ball  of  the 
finger;  the  razor  is  then  dipped  into  spirit,  and  as 
thin  a  slice  as  possible  (it  need  not  be  very  large)  is 
removed  from  the  inner  surface  of  the  bloodvessel 
and  placed  in  the  spirit,  after  which  one  or  two 


THE    LARGER    BLOODVESSELS.  139 

more  may  be  taken  from  other  parts.  In  making 
sections  when  the  piece  is  held  in  this  way,  it  will 
be  found  convenient  to  cut  from  the  operator. 

The  slices  are  taken  up  on  a  needle  or  section-lifter 
and  placed  on  a  slide  with  the  stained  surface  upper- 
most ;  they  should  be  in  as  little  spirit  as  possible, 
but  at  the  same  time  should  not  be  allowed  to  be- 
come actually  dry.  A  drop  of  the  ordinary  glycerine 
solution  is  placed  on  a  cover-glass,  and  this  is  quickly 
inverted  over  the  sections.  On  examinig  them  with 
the  microscope,  the  outlines  of  the  epithelioid  cells, 
and  perhaps  also  their  nuclei,  will  be  seen  in  those 
sections  which  were  made  from  the  gray  part  of  the 
bloodvessel.  In  sections,  however,  which  include 
any  of  the  patches  which  look  brown  to  the  naked 
eye,  it  will  be  found  that  the  difference  of  color  is 
due  to  the  epithelioid  cells  having  at  these  parts 
become  accidentally  rubbed  or  washed  away  before 
the  silver  solution  was  allowed  to  act ;  for  since  the 
subjacent  tissues  (the  sub-epithelial  connective  tissue, 
if  present,  arid  the  muscular  tissue  of  the  middle 
coat)  contain  more  intercellular  or  ground-substance 
than  the  epithelioid  layer  (where  it  only  occurs  in 
tine  lines  between  the  cells),  they  assume  a  browner 
appearance  after  the  reduction  of  the  silver,  and 
show  under  the  microscope  in  the  one  "case  irregular 
white  patches — the  cell  spaces — upon  the  brown 
ground,  and  in  the  other  transversely  arranged 
lanceolate  white  markings — the  plain  muscular  fibre 
cells — with  a  variable  amount  of  ground-substance 
between.  The  latter  appearance  may  be  obtained  all 
over  the  preparation  if  the  bloodvessel  which  is  to 
be  treated  with  silver  nitrate  is  first  brushed  with 
a  camel-hair  pencil  moistened  with  distilled  water, 
for  by  this  means  the  epithelioid  cells  are  removed, 
and  also,  for  the  most  part,  the  sub-epithelial  con- 
nective tissue  Avhere  present,  the  elastic  layer  being 
the  only  part  of  the  internal  coat  which  remains, 
and  since  this  does  not  reduce  the  silver  salt  the 
muscular  laver  is  the  one  which  is  seen  in  such  cases. 


140  PRACTICAL    HISTOLOGY. 

Except  when  the  epithelioid  cells  are  first  removed, 
either  purposely  or  accidentally,  even  a  compara- 
tively long  exposure  to  the  action  of  the  nitrate  of 
silver  solution  will  not  cause  the  deeper  coats  to 
become  stained.  This  is  the  case  with  all  structures 
which  are  coated  with  epithelioid  cells. 

Preparation  2.  Elastic  layers  ;  fenestrated 
membrane ;  muscular  tissue. — To  prepare  these 
several  parts,  a  piece  of  artery  (or  vein)  is  taken  (as 
fresh  as  possible,  but  this  is  not  so  imperative  as  for 
the  preparation  of  the  epithelioid  layer),  and  placed 
for  a  week  or  more  in  a  weak  solution  of  bichromate 
of  potash  (about  1  in  800),  the  fluid  being  changed 
every  other  day.  The  piece  is  then  taken  out,  pinned 
down  on  a  cork,  with  the  inner  surface  uppermost, 
and  a  thin  strip  torn  off  from  the  inner  surface  with 
fine  forceps.  This  is  transferred  to  a  slide,  and  teased 
as  finely  as  possible  in  a  drop  of  water.  It  will  be 
found  advantageous  to  employ  only  as  much  water 
as  will  keep  the  tissue  moist,  and  to  add  more  of 
this  by  placing  a  drop  on  the  cover-glass  before  it  is 
laid  on.  If  the  small  pieces  are  then  examined,  it 
will  be  found  that  they  are  for  the  most  part  made 
up  of  a  close  network  of  elastic  fibres  of  varying 
degrees  of  fineness.  Many  of  them  have  very  broad 
fibres  and  small  meshes,  so  that  there  may  be  found 
in  different  arteries  every  transition  to  the  true  elastic 
membrane.  This  will  itself  in  all  probability  be  met 
with  projecting  at  the  edges  of  some  of  the  fragments 
of  tissue,  or  even  entirely  separated  ;  the  fragments 
are  generally  curled  at  the  edges,  are  often  striated, 
and  nearly  always  exhibit  rounded  holes.  These 
fenestrated  membranes  are  more  frequently  met  with 
in  the  inner  coat  of  the  smaller  or  medium  sized 
arteries  (such  as  the  basilar),  than  in  the  largest  ves- 
sels (such  as  the  aorta). 

But  besides  the  different  kinds  of  elastic  tissue 
there  is  also  to  be  found  in  nearly  every  such  prepa- 
ration a  number  of  plain  muscular  fibre-cells  scattered 
about  in  the  fluid;  for,  in  stripping  off'  the  inner 


THE    LARGER    BLOODVESSELS.  141 

coat,  shreds  of  the  middle  coat  nearly  always  adhere 
to  it,  and  the  muscular  cells  of  this  readily  separate 
after  the  maceration  in  bichromate.  But  the  isolated 
cells  present  in  most  instances  such  a  ragged  shape- 
less aspect  that  they  would  hardly  be  known  for 
muscular  tissue.  A  convincing  proof,  however,  is 
the  addition  of  a  little  weak  logwood  solution  at 
the  edge  of  the  cover-glass.  This,  as  it  comes  to 
each  of  the  cells  in  question,  almost  instantaneously 
stains  their  long  rod-shaped  nuclei  of  an  intense 
violet,  whilst  the  body  of  the  cell,  if  the  logwood 
solution  be  sufficiently  weak,  remains  uncolored. 
The  addition  of  a  little  glycerine  at  the  edge  of  the 
cover-glass,  and  the  subsequent  cementing,  are  suffi- 
cient to  preserve  the  preparation. 

The  connective  tissue  and  elastic  fibres  of  the  outer 
coat  (as  well  as  the  muscular  tissue  of  the  same  coat 
in  certain  veins)  can  be  equally  well  seen  in  a  teased- 
out  preparation. 

Preparation  3.  Study  of  the  structure  of  the 
bloodvessels  by  means  of  sections. — To  form  a 
correct  idea  of  the  relative  thickness  of  the  several 
coats,  as  well  as  to  observe  the  differences  in  arrange- 
ment in  different  arteries  and  veins,  it  is  necessary 
to  study  them  in  vertical  sections,  f.^.,  sections  made 
in  a  direction  at  right  angles  to  their  surface.  Such 
sections  may  be  either  transverse  or  longitudinal ;  it 
will  be  better  perhaps  to  choose  the  former  direction, 
for  the  middle  coat  is  thereby  better  displayed.  But 
the  vessel  to  be  cut  must  tirst  be  hardened.  This 
may  be  effected  speedily  by  immersing  it  in  very 
strong  spirit  for  a  day  or  two,  and  indeed  this  method 
can  be  employed  tor  nearly  all  the  tissues  and  organs. 
It  is  preferable,  however,  in  many  instances  to  effect 
the  process  more  slowly,  by  means  of  some  watery 
fluid,  such  as  a  strong  solution  of  bichromate  of 
potash  or  a  weak  solution  of  chromic  acid,  since  in 
this  way  the  parts  shrink  less  and  consequently 
retain  their  form  better;  the  process  should,  never- 
theless, always  be  completed  by  means  of  spirit.  In 


142  PRACTICAL     HISTOLOGY. 

the  case  of  the  bloodvessels  an  immersion  for  a  fort- 
night or  three  weeks  in  a  1  per  cent,  solution  of 
bichromate  of  potash  answers  best ;  the  pieces  are 
then  placed  for  a  day  in  weak  spirit,  and  finally 
transferred  to  strong  methylated  alcohol.  Here  they 
may  remain  without  detriment  until  it  is  convenient 
to  prepare  the  sections. 

The  method  of  cutting  and  preparing  sections  for 
the  microscope  is  in  the  main  the  same  for  nearly  all 
the  tissues  and  organs  of  the  body,  and  since,  in  the 
further  study  of  these,  it  will  be  in  almost  constant 
requisition,  it  will  be  convenient  to  give  in  this 
place  an  account  of  the  mode  usually  adopted,  re- 
serving any  special  modifications  which  it  may  be 
needful  to  make  in  the  preparations  of  particular 
organs  until  these  come  to  be  treated  of. 


METHOD  OF  PREPARING  SECTIONS. 

Preparation  of  an  embedding  mass.— Unless 
the  piece  of  tissue  is  large  enough  to  admit  of  being 
held  firmly  by  the  left  hand  while  sections  are  made 
with  a  razor  held  in  the  right  hand,  it  is  necessary 
to  support  it  in  some  way.  This  is  usually  done  by 
surrounding  it  in  a  mould  with  some  fatty  mixture 
which  requires  only  a  moderate  temperature  to  melt 
it;  the  mixture  or  mass  should,  when  cold,  be  of 
about  the  same  consistence  as  the  hardened  tissue. 
Perhaps  the  most  generally  useful  mixture  for  this 
purpose  is  one  of  wax  and  olive  oil.  This  can,  by 
varying  the  proportions  of  the  ingredients,  be  suited 
at  will  to  the  consistence  of  the  tissue  which  it  is 
desired  to  obtain  sections  of.  For  most  purposes  the 
following  is  the  best  proportion  to  adopt,  and  it  is  to 
be  observed  that  whenever  subsequently  the  "wax- 
mass"  is  mentioned,  this  particular  mixture  is  indi- 
cated :  Sixty  cubic  centimetres  of  olive  oil  are  poured 
into  a  measuring-glass,  and  small  lumps  of  white  wax 
are  added  to  the  fluid  until  it  occupies  a  hundred 
cubic  centimetres.  It  is  now  placed  in  a  porcelain 


METHOD    OF    EMBEDDING. 


143 


or  tin  vessel  (a  small  oil-can  answers  the  purpose 
very  well),  and  heated  over  a  water-bath  until  the 
wax  is  entirely  melted.  The  mixture  is  then  thor- 
oughly stirred,  and  put  aside  until  wanted.  In 
laboratories  where  it  is  likely  to  be  much  in  requisi- 
tion it  is  well  to  have  a  supply  always  ready  melted. 
This  may  be  done  by  keeping  it  in  a  sandbath  at  a 
temperature  of  about  40°  C. 

Process  of  embedding, — One  or  more  shallow 
oblong  moulds  of  different  sizes  should  be  kept  in 
readiness  for  embedding  pieces  of  tissue  in.  They 
may  be  made  of  thin  pliable  metal,  such  as  sheet 
lead  or  capsule  metal  (Fig.  23),  so  as  to  permit  the 
sides  to  be  readily  bent  back  and  the  wax-mass 
removed  when  set;  but  if  these  are  not  at  hand  a 
suitable  mould  may  always  be  extemporized  out  of 
ordinary  stiff  paper.  A  piece  is  cut  of  about  the 
size  and  shape  shown  in  the  accompanying  diagram, 
Fig.  21,  and  folded  along  the  lines  there  marked  out, 

Fig.  21. 


Outline  showing  the  manner  in  which  a  small  piece  of  paper  is  to  be 
folded  to  make  an  embedding  trough. 

the  small  dotted  diagonals  at  the  corners  being  scored 
with  some  blunt-pointed  instrument,  an  ordinary 
hard  lead  pencil,  for  instance.  These  corners  are 
then  pinched  up  between  the  finger  and  thumb, 
bent  round  so  as  to  be  applied  to  the  ends  of  the 


144 


PRACTICAL    HISTOLOGY. 


oblong  (Fig.  22,  a),  and  are  fixed  there  by  turning 
down  the  flaps  cc  (Fig.  22,  b). 

Fig.  22. 


Embedding  trough  made  from  a  piece  of  piper  of  the  size  shown  in  Fig. 
21,  with  one  side,  b,  completed;  the  other,  «,  only  half  finished ;  so  as 
to  show  the  manner  in  which  the  corners  are  folded  and  fixed. 

The  trough  being  ready,  it  is  placed  upon  a  flat 
cork,  and  the  next  thing  is  to  take  the  piece  of  tissue 
(artery,  for  example)  out  of  spirit  and  place  it  for  a 
minute  or  two  on  clean  blotting-paper  to  remove  the 
excess  of  fluid;  at  the  same  time  its  surfaces  should 

Fig.  23. 


Embedding  trough  of  capsule  metal  placed  on  a  cork,  and  with  a  piece  of 
tissue  (artery)  in  situ  All  that  is  further  necessary  is  to  fill  the  trough 
with  melted  wax-mass. 

on  no  account  be  allowed  to  become  actually  desic- 
cated. A  minnikin  pin  is  then  pii^s-jd  for  a  good 
part  of  its  length  through  the  piece  near  the  end 
furthest  from  that  which  it  is  desired  to  cut,  and 


METHOD    OF    CUTTING    SECTIONS.  145 

the  point  of  the  pin  is  stuck  through  the  bottom  of 
the  mould  into  the  cork  in  such  a  way  that  the 
tissue  is  placed  at  one  end  of  the  oblong,  and  sus- 
tained in  a  horizontal  position  about  half  way  down 
in  the  embedding  trough,  as  shown  in  Fig.  23.  All 
is  done  as  quickly  as  possible,  so  as  not  to  allow  the 
tissue  time  to  become  too  dry  (if  there  is  any  fear  of 
this  it  must  be  moistened  now  and  then  with  a  drop  of 
spirit),  and  without  manipulating  the  piece  more  than 
can  possibly  be  helped.  The  object  to  be  cut  being 
thus  fixed  in  the  proper  position,  melted  wax-mass 
is  poured  into  the  mould  so  as  to  fill  it  completely, 
and  entirely  to  surround  the  piece  of  tissue,  and  the 
mould  is  set  aside  to  cool.  It  is  important  not  to 
use  the  mass  hotter  than  just  above  its  melting-point, 
as  the  tissues,  even  when  hardened,  are  liable  to  be 
injured  by  too  high  a  temperature.  When  the  wax 
is  completely  set  the  pin  is  carefully  withdrawn  and 
the  trough  removed  from  the  cork  and  placed  in 
spirit.  Here  the  cooling  is  soon  completed,  and  the 
paper  or  capsule  metal  having  been  taken  away  the 
embedded  tissue  is  ready  for  cutting. 

Method  of  cutting  the  sections. — The  sections, 
unless  a  microtome  or  section  instrument  is  used, 
when  a  special  form  of  knife  is  often  provided,  are 
made  with  an  ordinary  razor,  the  blade  of  which 
should  be  somewhat  hollow-ground  on  both  surfaces. 
Some  prefer  that  the  under  surface  should  be  ground 
flat,  and  this  is  advantageous  for  large  pieces  of 
tissue,  but  it  is  not  so  readily  kept  sharp.  For  this 
last- mentioned  purpose  a  good  strop  is  absolutely 
necessary.  The  razor  is  kept  constantly  wetted 
during  use  with  spirit,  contained  in  a  lar^e  shallow 
glass  dish,  into  which  the  instrument  is  dipped  from 
time  to  time,  and  in  which  also  the  sections  are  in 
the  first  instance  placed. 

All  then  being  ready,  the  cake  of  wax-mass  (pro- 
tected from  the  warm  fingers  by  being  wrapped 
round  with  a  small  strip  of  bibulous  paper)  is  held 
in  the  left  hand  with  the  end  in  which  the  tissue 
13 


146 


PRACTICAL    HISTOLOGY 


lies  uppermost,  whilst  the  razor  is  held  in  the  way 
shown  in  Fig.  24,  and  the  wax-mass  is  evenly  and 
gradually  shaved  off  layer  by  layer  until  the  em- 
bedded tissue  conies  into  view.  The  pieces  of  wax- 
Fig.  24. 


Process  of  cutting  sections  of  the  embedded  tissue. 

w,  cake  of  wax-mass,  with  the  piece  of  tissue  seen  in  it  as  a  dark  patch,  com- 
pletely inclosed  and  supported  by  the  wax  ;  p,  bibulous  paper  wrapped 
round  the  wax-mass,  except  where  this  is  being  cut  ;  d,  dish  containing 
spirit;  c,  glass  cover  with  section  lifter  lying  upon  it.  The  flask  on  the 
right  of  the  figure  serves  to  receive  the  spirit  which  has  been  used,  for  this 
may  be  employed  again  and  again  if  filtered  back  after  use. 

mass  which  are  removed  in  this  way  must  not  be 
allowed  to  fall  into  the  spirit,  but  are  to  be  put  on 
one  side,  and  the  razor  wiped  clean.  It  is  then  again 
dipped  in  the  spirit,  and  a  fresh  layer  of  the  cake 
removed,  including  a  moderately  thin  section  of  the 
tissue.  This  is  not  to  be  kept,  the  only  object  in 
making  it  having  been  to  prepare  a  clean  even  sur- 
face to  cut  from.  The  razor  is  again  wiped  clean  — 
this  must  be  done  before  each  fresh  section — re- 
wetted  with  spirit,  and,  starting  at  the  edge  of  the 


MODE    OF    CUTTING    SECTIONS.  147 

piece  of  tissue,  a  section  as  thin  as  possible  is  made, 
and  removed  on  the  razor  blade  without  bringing 
away  more  of  the  wax-mass  than  can  be  helped. 
The  section,  if  it  seems  thin  enough,  is  floated  off 
into  the  dish  of  spirit,  and  another  and  another  are 
made  in  the  same  manner.  No  difficulty  will  be 
experienced  in  getting  rid  of  any  wax  that  may  be 
still  adherent  to  the  sections  ;  a  touch  with  a  needle 
is  generally  sufficient. 

The  attainment  of  dexterity  in  cutting  thin  sections  in 
this  manner  with  the  free  hand — that  is  to  say,  without 
the  aid  of  any  special  instrument — is  in  great  measure,  as 
may  well  be  supposed,  a  matter  of  practice.  However, 
one  or  two  hints  drawn  from  experience  may  not  be 
superfluous. 

In  the  first  place  the  razor  must  be  always  kept  very 
sharp:  this  is  the  main  secret  of  thin  sections.  It  should 
also,  at  the  moment  of  cutting,  always  have  a  flood  of 
spirit  upon  the  upper  surface.  The  object  of  this  is  to 
keep  the  section  free  as  it  is  being  made,  otherwise  it  is 
apt  to  adhere  to  the  razor  and  to  become  broken.  Then, 
in  cutting,  the  razor  must  not  simply  be  pushed  forward 
over  the  tissue,  but  drawn  over  it  with  a  movement  from 
heel  to  point ;  but  at  the  same  time  the  whole  section 
must  be  completed  with  one  such  sweep,  for  a  to-and- 
fro  movement  inevitably  produces  ridges  upon  the  sur- 
face of  the  section.  Except  in  the  case  of  injected  pre- 
parations, the  sections  can  hardly  be  cut  too  thin ;  as  a 
rule,  those  sections  only  are  worth  keeping,  which  can 
barely  be  seen  as  they  are  being  cut.  It  must  not  be 
forgotten  that  they  will  appear  considerably  thicker  after 
they  are  stained. 

Two  or  three  sufficiently  thin  sections  having  been 
in  this  way  obtained,  the  embedded  tissue  may  be 
put  aside  in  spirit  for  the  present,  in  case  any  more 
should  subsequently  he  required. 

The  sections  have,  in  the  tirst  place,  to  be  stained. 
This  is  best  effected,  in  the  case  of  the  sections  of 
artery,  by  immersing  them  in  the  ordinary  logwood 
alum  solution  for  a  few  minutes.  They  are  then 
put  into  water  for  a  second  or  two,  to  wash  away 


148 


PRACTICAL    HISTOLOGY. 


the  excess  of  staining  fluid,  which  would  otherwise 
he  precipitated,  and  are  transferred  from  this  to 
spirit  (absolute  alcohol  is  gene- 
Fig'-  25-  rally  recommended,  but  strong 
methylated  spirit  is  sufficiently 
anhydrous)  in  which  they  are 
allowed  to  lie  until  it  is  certain 
that  all  the  water  is  replaced 
(live  minutes  is  generally  amply 
sufficient),  after  which  they  are 
placed  in  oil  of  cloves  for  a 
minute  or  two.  In  this  the 
sections  should  almost  imme- 
diately become  clear  and  trans- 
parent, and  the  change  has  at 
the  same  time  the  effect  of  caus- 
ing their  color  to  appear  darker. 
If  any  parts  of  the  section  do 
not  participate  in  the  change  of 
appearance,  but  remain  opaque 
and  cloudy,  it  is  a  sign  that  all 
the  water  has  not  been  extracted, 
either  because  the  sections  were 
not  left  long  enough  in  spirit,  or 
because  this  was  not  anhydrous 
enough  (for  oil  of  cloves  will  not 
mix  with  water  nor  with  alcohol 
which  contains  more  than  a  trace 
of  water).  But  it  is  not  too  late 
to  put  them  from  the  oil  into 
strong  spirit  again,  and  when 
they  have  been  long  enough 
there  to  transfer  them  once  more 
to  oil  of  cloves.  All  these  trans- 
ferences may  be  effected  by  aid 
of  a  section-lifter  (Fig.  25),  made 
from  a  piece  of  copper  or  i>'ernian- 

Section-lifter.    Natural  *  . 

size.  silver  wire  flattened  out  at  one 

or    both    ends,   which    are    bent 

over  so  as  to  be  hoe-shaped.     l>y  means  of  this  in- 


TRANSFERENCE    OF    SECTIONS.  149 

strument  even  very  thin  arid  fragile  sections  may  be 
readily  lifted  from  one  vessel  to  another  without 
injury.  The  staining  fluid,  which  should  always  be 
freshly  filtered,  and  not  be  used  for  too  many  sec- 
tions, may  be  in  a  watch-glass,  which  should  be 
covered  over  by  another  watch-glass  inverted,  but 
the  other  fluids  which  are  constantly  in  use  it  is 
better  to  keep  in  covered  glass  or  china  pots,  for 
they — especially  the  oil  of  cloves — may  be  used  for 
a  considerable  number  of  sections  without  detriment. 
The  process  of  preparation  may  be  arrested  at  any 
stage  and  completed  at  another  time,  if  the  sections 
are  left  in  spirit  or  in  oil  of  cloves. 

In  transferring  sections* from  one  fluid  to  another, 
and  especially  when  they  are  put  in  the  staining 
fluid,  care  should  be  taken  that  they  are  actually 
immersed,  and  do  not  merely  float  on  the  surface. 

Instead  of  the  ordinary  logwood  solution  Kleinen- 
berg's  fluid  (see  post)  may  with  advantage  be  used 
for  staining  sections  that  are  to  be  mounted  in  bal- 
sam or  dammar.  In  this  case  the  sections  can  be 
placed  from  spirit  into  the  staining  fluid,  and  from 
the  latter  directly  into  the  spirit  again  without  the 
necessity  for  placing  them  in  water  at  all. 

The  sections  then  being  in  oil  of  cloves,  and  the 
clearing  up  satisfactorily  effected,  all  that  remains 
is  to  mount  them  permanently.  For  this  purpose 
either  dammar  varnish  or  a  solution  of  Canada  bal- 
sam in  chloroform — the  same  liquid  that  was  em- 
ployed for  cementing  the  cover-glass  of  the  glycerine 
preparations — is  employed.1  These  solutions,  al- 
though perfectly  limpid  even  in  the  cold,  readily 
dry  when  exposed  to  the  air,  and  thus  become  hard 

1  The  Canada  balsam  used,  must  be  heated  over  a  sandbath 
until  it  is  quite  hard  when  cold  ;  it  is  then  dissolved  in  an  equal 
volume  of  chloroform.  Dammar  varnish  is  made  by  dissolving  one 
part  of  gum  dammar  in  two  parts  of  oil  of  turpentine,  and  one  part 
of  gum  mastic  in  two  parts  of  chloroform  ;  mixing  the  solutions 
and  filtering.  The  mixture  is  apt  to  become  opalescent  on  keep- 
ing, but  this  disappears  permanently  on  boiling  the  fluid. 

13*  ' 


150  PRACTICAL    HISTOLOGY. 

and  firm.  The  sections  are  removed  by  the  lifter 
from  the  oil  of  cloves  and  gently  placed  upon  a  slide, 
the  excess  of  oil  of  cloves  is  allowed  to  drain  off  or 
is  soaked  up  with  blotting  paper,  a  drop  of  the 
mounting  varnish  is  placed  on  the  cover-glass,  and 
this  is  then  quickly  inverted  over  the  sections.  The 
preparation  is  now  complete  and  permanent,  and 
ready  for  examination  ;  but  as  it  will  be  some  while 
before  the  mounting  varnish  has  become  hard  to  any 
distance  under  the  cover-glass,  care  should  be  taken 
not  to  let  this  become  shifted  in  any  way,  since  such 
treatment  would  probably  crumple  or  otherwise 
spoil  the  sections. 


Preparation  4.  The  smaller  arteries  and 
veins  and  the  capillaries. — The  elongated  epi- 
thelioid  cells  which  form  the  walls  of  the  capillaries, 
and  which  line  the  arteries  and  veins,  are  readily 
shown  in  silvered  preparations  of  any  vascular 
tissue.  It  is  most  convenient  to  choose  a  vascular 
membrane  for  the  purpose,  because  more  readily  dis- 
played, and  of  vascular  membranes  the  mesentery 
of  the  frog  or  toad  is  perhaps  as  easy  to  prepare  as 
another.  The  following  is  the  mode  of  proceeding: 
The  animal  (a  male)  having  been  decapitated  and 
the  spinal  cord  destroyed,  the  trunk  is  suspended 
for  a  few  minutes  by  the  lower  limbs  in  order  to 
allow  the  blood  to  drain  from  the  body  as  completely 
as  possible.  The  frog  is  then  placed  on  its  back, 
and  the  abdomen  freely  opened.  A  loop  of  intes- 
tine is  seized  with  forceps  and  gently  raised  by  an 
assistant,  while  with  a  soft  camel-hair  pencil,  mois- 
tened with  distilled  water,  the  operator  carefully 
brushes  the  mesentery  on  both  surfaces,  carrying  the 
brush  in  every  case  from  the  intestine,  not  towrards 
it.  This  brushing  subserves  two  purposes — in  the 
first  place,  the  epithelioid  cells  of  the  serous  mem- 
brane, which  would  obstruct  the  passage  of  the 
silver  solution  to  the  bloodvessels, are  removed  ;  and 


SMALLER    BLOODVESSELS.  151 

in  the  second  place,  much  of  the  blood  which 
remains  in  the  vessels  is  driven  out  of  them. 

The  brushing  being  completed,  the  loop  of  intes- 
tine, with  its  included  mesentery,  is  cut  off,  dipped 
for  a  second  into  a  capsule  of  distilled  water,  and 
then  at  once  placed  in  J  per  cent,  nitrate  of  silver 
solution.  Here  it  is  allowed  to  remain  ten  minutes  ; 
after  which  it  is  rinsed  in  distilled  water  and  then 
placed  in  common  water  in  the  sunlight.  If  the 
day  is  bright,  the  silver  is  soon  reduced,  and  all 
that  remains  to  be  done  is  to  place  the  preparation  in 
a  shallow  glass  dish,  and  carefully  cut  off  and  remove 
the  piece  of  intestine,  leaving  the  mesentery.  This 
must  be  done  under  water,  and  will  require  sharp 
scissors  and  delicate  handling,  so  as  not  to  drag 
upon  the  mesentery  or  throw  it  into  folds. 

In  order  to  mount  it  a  slide  is  held  in  the  water 
and  the  membrane  allowed  to  float  over,  after  which 
the  slide  is  carefully  lifted  out  with  the  membrane 
flat  upon  it,  and  the  excess  of  water  is  drained  off. 
Before  covering  it  the  preparation  must  be  examined, 
both  with  the  unassisted  eye  and  with  a  low  power 
of  the  microscope,  so  as  to  detect  any  folds  or  creases 
in  it.  If  present  they  can  be  got  rid  of,  by  gently 
drawing  out  first  this  corner  and  then  that  with  a 
needle.  A  drop  of  strong  glycerine  may  then  be 
placed  on  the  middle  of  the  preparation,  and  the 
cover-glass  laid  on  and  allowed  slowly  to  settle 
down.  More  glycerine  may  be  afterwards  added  at 
the  edge  if  necessary.  Should  it  be  found,  on  exam- 
ining the  preparation  with  a  moderately  high  power, 
that  the  outlines  of  the  epithelioid  cells  of  the  vessels 
are  not  yet  sufficiently  marked,  it  will  be  well  to 
leave  the  preparation  out  in  the  light,  but  covered 
over  from  dust,  for  a  day  or  two. 

If  there  is  but  little  sunlight  the  reduction  of  the 
silver  may  often  be  better  effected  by  placing  the 
loop  of  intestine,  with  its  attached  mesentery,  after 
it  has  been  taken  from  the  silver  solution  and  rinsed 
in  water,  in  a  beaker  of  weak  spirit  (equal  parts  of 


152  PRACTICAL    HISTOLOGY. 

water  and  strong  spirit,  freshly  prepared),  and  expos- 
ing it  to  the  light  in  this  for  an  hour  or  more.  The 
cutting  off  of  the  intestine  must  be  performed  in  the 
same  fluid  and  the  mesentery  floated  from  it  on  to 
the  slide.  The  method  has  the  advantage  not  only 
of  effecting  the  reduction  of  the  metal  with  greater 
surety,  but  also  of  rendering  it  easy  to  obtain  the 
membrane  free  from  creases,  for  the  mesentery  is 
partly  hardened  by  the  spirit  while  in  a  state  of 
extension,  and  continues  in  this  condition  when 
floated  on  to  the  slide,  so*that  it  is  seldom  necessary 
further  to  extend  it  by  artificial  stretching.  In 
these  silvered  preparations  little  but  the  epithelioid 
cells  can  be  made  out,  for  the  rest  of  the  tissue 
generally  remains  almost  unstained,  and  becomes 
very  transparent  in  glycerine. 

Preparation  5. — To  exhibit  the  muscular  struc- 
ture of  the  small  arteries  and  veins,  and  the  nuclei 
of  their  epithelioid  lining  and  of  the  walls  of  the 
capillaries,  the  vessels  are  stained  with  logwood. 
This  is  done  by  immersing  the  mesentery  or  other 
vascular  membrane,  either  fresh,  or  better  after  hav- 
ing lain  for  a  day  or  two  in  very  weak  bichromate 
of  potash  solution,  in  a  dilute  solution  of  logwood 
alum,  until  distinctly  colored;  then  place  the  tissue 
in  water,  and  mount  it,  with  the  same  precautions 
as  before  to  prevent  creasing,  in  glycerine.  For  the 
structure  of  the  small  arteries  the  pia  mater  from 
the  human  brain  may  be  used.  A  small  piece  is 
stripped  off  with  forceps ;  and  as  it  consists  almost 
entirely  of  small  arteries  and  veins,  and  moreover 
a  few  capillaries  are  generally  dragged  out  with,  it 
from  the  cerebral  substance,  the  structure  of  all  the 
vessels  is,  after  staining,  very  well  displayed.  The 
small  veins  are  here  exceptional  in  being  entirely 
devoid  of  a  muscular  coat,  whereas  the  arteries  have 
this  coat  well  developed,  and  it  is  particularly  well 
shown  in  consequence  of  the  staining  of  the  trans- 
verse nuclei  of  the  muscular  fibres  by  the  logwood. 
Within  these  Tnay  be  detected,  by  carefully  using 


STUDY    OF    THE    CIRCULATION.  153 

the  fine  adjustment  of  the  microscope,  in  the  first 
place,  longitudinal  striae,  which  are  produced  by 
wrinklings  in  the  elastic  layer  of  the  internal  coat ; 
and  in  the  second  place,  situated  most  internally, 
elongated  oval  nuclei,  which  belong  to  the  epithe- 
lioid  cells  lining  the  vessel.  Of  course,  two  layers 
of  all  these  structures  are  come  across  in  focussing 
from  above  down.  The  outer  coat  is  represented 
merely  by  a  few  corpuscles  and  .fibres  of  connective 
tissue  which  blend  externally  with  the  connective 
tissue  framework  of  the  membrane. 

It  will  be  found  in  carefully  focussing  from  above 
down,  that  at  one  position  of  the  focus  tlie  small 
vessel  has  exactly  an  appearance  as  if  it  had  been 
cut  longitudinally  through  the  middle,  and  as  if  the 
top  of  the  lower  half  were  being  examined.  The 
lumen  is  seen  in  the  centre,  with  possibly  a  few 
blood  corpuscles  still  in  it ;  on  either  side  of  this  a 
well-marked  line  representing  the  inner  coat ;  out- 
side this  again  what  seems  like  a  row  of  rounded 
cells,  which  are  really  the  encircling  fibre-cells  of 
the  muscular  coat  seen  as  if  cut  across;  and  finally, 
here  and  there,  outside  of  all,  small  cells  presenting 
the  fusiform  aspect  of  connective  tissue  corpuscles 
seen  in  profile.  All  these  appearances  are  exactly 
the  same  as  if  a  section  had  been  made  along  the 
vessel,  and  result  from  the  fact  that  only  those  parts 
of  an  object  which  lie  in  the  horizontal  plane  that 
happens  to  coincide  with  the  focal  distance  of  the 
objective,  are  distinctly  seen,  so  that  it  seems  as  if 
only  this  particular  slice  tvere  present.  An  "  optical 
longitudinal  section"  is  thus  obtained  of  the  vessel. 

STUDY  OF  THE  CIRCULATION. 

The  study  of  the  bloodvessels  cannot  be  said  to 
be  in  any  s.ense  of  the  word  complete  until  they  have 
been  viewed  in  the  living  condition  and  with  the 
blood  still  moving  through  them.  Such  an  observa- 
tion can,  of  course,  only  be  made  whilst  an  animal 


154  PRACTICAL    HISTOLOGY. 

is  still  alive,  and  in  parts  which  are  transparent 
enough  to  allow  the  vessels  to  be  distinctly  seen. 
Membranous  parts  are  those  which  are  naturally 
best  adapted  for  such  observation,  as,  for  example, 
the  web  of  the  frog's  foot  and  of  the  bat's  wing  ; 
the  tongue,  mesentery,  and  lungs  of  the  frog  and 
toad,  but  especially  the  latter  animal ;  and  the 
mesentery  and  omentum  of  small  mammals.  In  such 
preparations  the  surrounding  tissues,  and  especially 
the  connective  tissue  corpuscles,  may  be  studied  as 
well  as  the  bloodvessels ;  and  the  changes  due  to 
commencing  inflammation  which  are  exhibited  by 
the  bloodvessels,  and  the  migration  from  the  veins 
of  the  white  blood  corpuscles,  can  always  be  brought 
on  either  by  the  application  of  irritants,  or,  as  in 
the  case  of  the  serous  membranes,  hy  simple  expo- 
sure to  the  air.  The  best  methods,  therefore,  of 
observing  the  circulation  of  the  blood  in  different 
parts  will  be  described  in  the  following  preparations. 
Preparation  6.  Circulation  in  the  frog's 
web. — One  of  the  common  English  frogs  (Rana 
temporaria),  of  as  light  a  color  as  possible,  is  chosen, 
and  by  means  of  a  Pravaz  or  other  hypodermic 
syringe  two  drops  of  a  very  weak  solution  of  curare 
(1  to  2000  of  water)  are  injected  under  the  skin  of 
the  back.  This  is  generally  sufficient,  in  the  course 
of  from  a  quarter  to  half  an  hour,  to  render  the 
animal  completely  motionless,  whilst  the  pulsations 
of  the  heart  and  the  circulation  proceed  unimpaired. 
The  frog  is  then  laid  on  a  piece  of  cork  or  soft  wood 
of  an  oblong  shape  which*  has  a  narrow  slit  at  one 
end.  One  of  the  interdigital  webs  is  placed  over 
this  slit  and  fastened  on  either  side  by  means  of  one 
or  two  minnikin  pins  or  fine  needle-points  passed 
through  the  adjoining  webs.  Care  must  be  taken 
that  the  web  under  examination  is  at  no  part  tightly 
stretched,  since  this  would  tend  to  arrest  -or  obstruct 
the  circulation.  A  slip  of  blotting-paper  or  a  piece 
of  linen  rag  is  placed  over  the  animal  and  kept 
thoroughly  wetted  with  water,  and  the  cork,  with 


CIRCULATION    IN    THE    MESENTERY.       155 

the  frog  upon  it,  is  then  placed  on  the  stage  of  the 
microscope  (the  head  of  the  animal  being  away  from 
the  observer,  and  the  web  over  the  aperture  in  the 
diaphragm),  and  is  fixed  in  this  position  by  the 
clamps  like  an  ordinary  slide.  To  observe  the  web 
a  low  power  is  used  to  see  the  general  features  of 
the  circulation,  a  high  power  being  afterwards  em- 
ployed to  observe  the  parts  more  in  detail.  But 
this  should  not  (unless  it  is  an  immersion)  be  of  too 
short  a  focal  distance,  since  otherwise  the  lower 
glass  is  apt  to  become  clouded  by  moisture  from  the 
web.  It  is  not  advisable  to  put  a  piece  of  covering 
glass  on  the  latter  to  prevent  the  clouding,  as  the 
circulation  might  thereby  be  interfered  with  or 
arrested. 

The  web  of  the  frog's  foot  is  the  easiest  of  the 
vascular  membranes  to  prepare,  but  has  the  disad- 
vantage that,  owing  to  its  comparative  thickness  and 
its  epidermic  covering,  it  is  not  always  easy  clearly 
to  make  out  the  intermediate  tissue.  At  the  same 
time,  being  under  almost  completely  natural  condi- 
tions, the  circulation  will  continue  for  an  indefinite 
time  quite  unimpaired. 

Preparation  7.  Circulation  in  the  mesen- 
tery.— The  mesentery  of  the  frog,  and  still  better 
of  the  toad,  is  admirably  adapted  by  its  thinness 
and  perfect  transparency,  as  well  as  it  great  vascu- 
larity,  for  observations  on  the  bloodvessels  and  sur- 
rounding tissues.  It  is  necessary  to  have  a  special 
mesentery-plate  for  this  purpose,  which  can,  how- 
ever, be  readily  made  from  an  oblong  piece  of  cork, 
like  that  used  for  the  web  observation.  A  round 
hole  about  half  or  three-quarters  of  an  inch  in 
diameter  is  made  at  one  side  with  a  cork  borer, 
and  a  small  piece  of  the  same  material  about  half 
an  inch  thick,  and  with  a  segment  of  a  similar  circle 
cut  out  of  its  side,  is  fixed  on  the  first  with  sealing- 
wax  or  small  pins  (Fig.  26,  b).  A  piece  of  glass  of  the 
same  shape  as  this  segment  may  be  fitted  into  it  near 
the  top  for  the  mesentery  to  rest  on.  The  animal — 


156  PRACTICAL    HISTOLOGY. 

a  male — having  been  rendered  insensible  by  destruc- 
tion of  the  brain,  or  other  means,  is  curarized  as 
before,  and  laid  upon  its  back,  and  a  longitudinal 
cut  about  an  inch  long:  is  made  with  scissors  through 

O  O 

Fig.  26. 


Flat  piece  of  cork  arranged  as  a  frog-stage  for  viewing  the  circulation 
in  the  web,  tongue,  mesentery,  or  lungs. 

Over  the  small  pieces  of  cork  a  the  tongue  cau  be  fixed  ;  n  can  he  removed  when 
the  slit  below  it  is  wauted  for  the  web  ;  b,  cork  with  a  deep  groove  cut  along 
one  side  ;  to  this  the  iute.sliae  is  fastened  by  needle-poiuts,  while  the  mesen- 
tery rests  on  a  semicircular  piece  of  glass  which  should  fit  at  the  top  of  the 
groove. 

the  skin  of  the  abdomen  about  half  an  inch  to  the 
right  of  the  middle  line.  Before  proceeding  further, 
the  operator  should  wait  for  a  minute  or  two  to 
make  sure  that  there  will  be  no  bleeding  ;  and  any 
blood  that  may  have  already  exuded  should  be  dried 
up  with  blotting-paper.  The  abdominal  cavity  is 
then  opened  by  a  corresponding  cut  through  the 
muscles  and  peritoneum,  taking  care,  however,  to 
avoid  any  veins  that  may  be  in  sight.  Having 
again  assured  himself  of  the  absence  of  bleeding, 
the  operator  very  gently  draws  out  one  of  the  coils 
of  the  intestine,  with  its  included  mesentery  at  the 
aperture.  The  animal  is  now  to  be  turned  over  on 
its  side,  and  so  propped  up  against  the  smaller  cork 
that  the  wound  is  about  on  a  level  with  the  top. 


CIRCULATION    IN    THE    MESENTERY,       157 

All  that  remains  to  be  done  is  to  place  the  extruded 
mesentery  over  the  aperture,  and  to  keep  it  in  posi- 
tion by  two  or  three  fine  needle  points  passed  through 
the  surrounding  intestine  into  the  cork.  In  this 
case  again,  the  greatest  care  must  be  taken  in  no 
way  to  drag  upon  the  exposed  membrane,  or  to 
allow  it  to  be  pressed  upon.  Moreover,  the  surface 
must  from  time  to  time  be  moistened  with  a  little 
salt  solution,  to  prevent  its  becoming  dry.  But  in 
spite  of  every  precaution  the  mere  exposure  of  the 
serous  surface  to  the  air  is  sufficient  to  produce 
before  long  the  changes  in  the  circulation  which 
are  characteristic  of  the  commencement  of  inflam- 
mation. 

Preparation     8.       Capillary     circulation     in 

Mammals. — It  is  less  easy  to  study  the  circulation  in 
the  serous  membrane  of  mammals,  for  the  exposure  re- 
quired for  the  purpose  is  apt  to  be  far  more  prejudicial  to 
the  maintenance  of  the  normal  condition  of  the  tissues 
than  is  the  case  with  the  cold-blooded  vertebrates.  It  is 
necessary,  moreover,  to  maintain  the  exposed  part  at  the 
body  temperature,  and  to  immerse  it  in  fluid,  since  it 
would  otherwise  become  at  that  temperature  rapidly 
desiccated.  The  membrane  generally  chosen  is  not  the 
mesentery  but  the  omentum,  which  in  many  animals,  e.g. 
the  guinea-pig,  is  very  extensive,  and  at  the  same  time 
thin,  and  provided  in  parts  with  a  sufficient  number  of 
bloodvessels.  The  animal,  whieh  should  be  rather  a 
small  one,  is  anaesthetized  with  chloral  hydrate,  a  few 
minims  of  a  50  per  cent,  solution  being  injected  under 
the  skin.  The  warm  stage  (Fig.  9)  is  in  the  meanwhile 
got  read}',  and  a  glass  tray  (which  can  be  extemporized 
out  of  a  small  plate  of  glass,  some  pieces  of  glass  rod  and 
sealing-wax)  is  placed  on  it  and  filled  with  salt  solution, 
which  is  maintained  at  about  38°  C.  Then  the  animal 
is  supported  on  a  block  at  a  convenient  level,  and  the 
abdomen  having  been  carefully  opened,  a  little  of  the 
omentum  is  drawn  out  and  allowed  to  float  flat  in  the 
warm  salt  solution,  where  it  can  be  examined  either  with 
a  low  power  or  with  an  immersion  objective  dipping  into 
the  solution.  If  the  latter  be  employed  a  piece  of  thin 
14 


158  PRACTICAL    HISTOLOGY. 

covering  glass  nnist  be  placed  over  the  part  of  the  mem- 
brane which  is  to  be  examined,  so  as  to  sink  it  in  the  fluid 
and  keep  it  steady.  But  in  spite  of  every  precaution,  the 
circulation  under  these  conditions  retains  its  normal 
character  but  a  short  while,  and  inflammatory  congestion 
and  stasis,  or  complete  stoppage  of  the  flow  of  blood, 
rapidly  supervenes.1 

Preparation  9.  Circulation  in  the  lung  of  the 
toad. — This  is  readily  observed  with  the  aid  of  the 
mesentery  board.  The  animal  must,  as  before,  be 
first  rendered  insensible  and  curarized,  but  it  will 
be  found  that  a  good-sized  toad  will  require  at  least 
six  times  as  much  curare  as  a  frog.  An  opening  is 
made  at  the  side  of  the  chest  .large  enough  to  allow 
the  lung,  which  in  the  toad  almost  always  remains 
distended  with  air,  to  protrude.  The  animal  is  then 
propped  up  on  the  mesentery  board  (Fig.  26)  in  such 
a  manner  that  the  lung  rests  over  the  aperture  ft, 
and  the  circulation  can  be  studied  in  the  part  which 
is  uppermost  without  further  trouble.  A  frog  may 
be  used  in  a  similar  way,  but  there  is  much  greater 
difficulty  in  keeping  the  lung  distended.  In  either 
case  the  greatest  care  must  be  taken  to  avoid  prick- 
ing, or  in  any  way  rupturing  the  wall  of  the  lung. 

Preparation  10.  Circulation  in  the  tongue 
of  the  toad. — By  far  the  most  beautiful  object  for 
studying  not  only  the  circulation  but  also  the  tissues 
in  the  living  animal,  is  the  tongue  of  the  toad,  and 
in  a  slightly  less  degree  that  of  the  frog.  The  tongue 
is  in  these  creatures  an  extremely  extensile  organ, 
which,  under  ordinary  circumstances,  lies  folded 
back  on  the  floor  of  the  mouth  (Fig.  27,  B),  but  which 
can  at  the  will  of  the  animal  be  protruded  for  a  con- 
siderable distance  (c).  For  the  preparation  of  the 
organ  the  cork  plate  is  again  necessary  ;  a  smaller 
piece  of  cork  of  the  shape  shown  in  the  figure  (Fig. 

1  For  a  detailed  account  of  this  method  the  student  is  referred 
to  the  original  description  by  Knrdon  Sanderson  and  Strieker, 
in  the  "  Quarterly  Microscopical  Journal"  for  1.870. 


CIRCULATION    IN    THE    TONGUE. 


159 


26,  a)  and  about  one-eighth  of  an  inch  thick,  being 
fastened  with  pins  over  the  slit  which  served  for  the 
display  of  the  frog's  web. 

Fig.  27. 


Structure  and  position  of  the  tongue  of  the  toad  (Dowdeswell). 

A.  Transverse  section  through  the  middle  of  the  organ  with  the  lymph-spaces 
fully  distended  ;  a  a,  thick,  papillated  mucous  membrane  ;  b,  tbin  lower 
membrane;  m,  muscular  bundle  cut  across,  united  to  the  sides  of  the  tongue 
by  septa  of  connective  tissue  8  ft;  v,  position  of  the  larger  bloodvessels. 

B    Profile  view  showing  the  tongue  in  its  ordinary  position  within  the  mouth. 

C.  The  same  when  extended. 

The  toad  having  been  rendered  insensible  and 
curarized  as  before  is  laid  upon  its  back  with  its 
nose  close  to  the  slit.  The  lower  jaw  is  then  raised 
and  the  folded-back  end  of  the  tongue  is  found,  and 
drawn  gently  out  of  the  mouth  with  forceps.  The 
end  has  a  pointed  projection  or  cornu  on  each  side ; 
these  are  successively  laid  hold  of  by  the  forceps. 


160  PRACTICAL    HISTOLOGY. 

and  fastened  with  needle  points  to  the  small  piece 
of  cork  on  either  side  of  the  slit.  Before  the  rest 
of  the  operation  is  described,  a  word  or  two  may  he 
said  with  regard  to  the  structure  of  the  organ.  It 
is  not  solid  throughout  as  in  mammals,  but  hollow, 
the  interior  being  occupied  by  a  lymphatic  cavity. 
This  lymph  space  is  traversed  by  bundles  of  muscu- 
lar fibres  (Fig.  27,  m)  which  pass  towards  the  ex- 
tremity of  the  organ  and  are  connected  to  the  sides 
by  delicate  septa  of  connective  tissue  (s  s).  Above 
the  lymph  space  in  the  present  position  of  the 
animal — on  its  back  with  the  tongue  extruded — the 
mucous  membrane  is  thick  and  papillated  (Fig.  27, 
A,  a  a).  Below  is  a  very  thin  and  delicate  mucous 
membrane  (/>),  with  numerous  bloodvessels,  and  small 
muscular  fibres  running  over  it.  The  former  mem- 
brane is  too  thick  and  irregular  to  allow  the  delicate 
internal  structures  to  be  seen  through  it ;  it  is  there- 
fore slit  up  longitudinally  and  pinned  to  either  side. 
But  to  do  this  without  injuring  tbe  delicate  parts 
below,  it  must  be  separated  from  them,  and  this  can 
best  be  effected  by  distending  the  lymph  sac  with 
salt  solution.  With  this  object  a  Pravaz  syringe 
(Fig.  17),  provided  with  a  fine  and  sharp  canula,  is 
filled  with  the  fluid,  and  its  point  is  stuck  into  the 
tongue  near  the  end,  passing  about  half  an  inch 
backwards.  It  will  almost  certainly  be  found  that 
on  pressing  the  piston  down,  the  salt  solution  will 
readily  flow  into  the  lyrnph  sac,  and  as  it  fills  this 
will  cause  the  thin  mucous  membrane  at  the  lower 
part  to  become  bagged  out,  and  completely  separated 
from  the  muscular  bundles,  m  m,  and  these  again 
from  the  thick  layer  above.  The  latter  is  now  care- 
fully slit  up  along  its  middle  by  sharp  fine  scissors, 
and  first  one  edge  of  the  wound  and  then  the  other 
is  drawn  to  the  side  of  the  slit  in  the  cork  and 
fastened  there  by  two  or  three  needle-points.  If 
everything  is  carefully  done,  there  will  be  no  escape 
of  blood  over  the  preparation  ;  but  should  any  blood 


OBSERVATION    OF    LIVING    TISSUES.        161 

have  exuded,  it  may  be  washed  off  by  pouring  a 
little  salt  solution  over  the  surface. 

There  is  now  brought  to  view  the  fan -like  group 
of  muscles  which  pass  through  the  middle  of  the 
lymph  sac,  and  the  bundles  of  which  are,  as  before 
mentioned,  connected  with  the  sides  and  with  one 
another  by  delicate  septa  of  connective  tissue,  tra- 
versed by  a  few  bloodvessels ;  and  it  is  this  delicate 
connective  tissue,  of  which  two  strata  can  generally 
be  traced,  one  superficial  to  the  other,  which  is  better 
adapted  than  any  other  part  for  exhibiting  both  ves- 
sels and  connective  tissue  corpuscles  (the  latter  rather 
peculiar  in  form  and  appearance).  Moreover,  the 
mere  exposure  of  the  lymphatic  surface  soon  causes 
inflammatory  changes,  and  after  the  preparation  has 
been  made  a  few  minutes  only,  the  first  commence- 
ment of  these  is  seen  in  the  sticking  of  the  pale  cor- 
puscles to  the  walls -of  the  vessels,  speedily  followed 
by  their  migration  from  the  veins  into  the  surround- 
ing tissue.  Nowhere  can  the  fact  be  more  clearly 
established,  and  the  details  of  the  process  more  accu- 
rately followed,  than  here.  Moreover,  if  the  animal 
is  kept  properly  moist  and  from  time  to  time  given 
an  additional  dose  of  curare,  it  remains  for  a  long 
while  in  a  perfectly  natural  condition,  and  the  in- 
flammatory process  can  be  studied  for  as  long  as  may 
be  desired.  By  examining  such  a  preparation  at 
intervals  during  several  days,  the  same  connective 
tissue  corpuscles  being  again  and  again  found  and 
brought  under  observation,  it  has  been  conclusively 
proved  that,  at  all  events  as  regards  these  corpuscles 
of  the  tongue  of  the  full-grown  toad,  the  connective 
tissue  corpuscles  take  no  active  part  in  the  process 
of  acute  inflammation.  The  circulation  of  the  blood 
among  the  muscular  flbres  can  also  be  well  seen  in 
this  part  of  the  tongue. 

Lastly,  by  focussing  through  the  connective  tissue 
septa,  or  by  severing  the  longitudinal  muscular  bun- 
dles which  they  serve  to  unite,  the  vessels  of  the 
lower  mucous  membrane  are  brought  into  view, 


162  PRACTICAL    HISTOLOGY. 

especially  if  a  slip  of  glass  is  fitted  into  the  small 
piece  of  cork,  so  as  to  support  the  tongue  and  pre- 
vent the  thin  membrane  from  bulging  downwards. 

METHOD  OF  INJECTING  THE  BLOODVESSELS. 

Before  leaving  the  subject  of  the  bloodvessels  the 
best  mode  of  filling  them  with  transparent  material 
may  be  described,  especially  as  in  the  study  of  the 
several  organs  it  is  necessary,  in  order  that  the  course 
and  arrangement  of  the  vessels  may  be  properly  made 
out,  that  sections  of  injected  as  well  as  of  uninjected 
preparations  should  be  looked  at.  It  will  be  conve- 
nient in  this  place  to  describe  the  injection  of  a  small 
animal  entire  from  the  aorta,  reserving  any  special 
directions  concerning  organs  which  are  not  thereby 
properly  injected,  such,  for  instance,  as  the  lungs  and 
liver,  until  they  are  severally  dealt  with. 

Preparation  of  the  injection  mass.— This  is 
almost  always  a  solution  of  gelatine  colored,  either 
red  with  finely  precipitated  carmine,  or  blue  with 
soluble  Prussian  blue.  Sometimes,  but  rarely,  when 
it  is  wished  to  inject  two  sets  of  vessels  of  different 
colors,  both  of  these  are  used,  but  as  a  rule  all  the 
bloodvessels — arteries,  capillaries,  and  veins — should 
be  filled  with  the  same  injecting  fluid;  preparations 
in  which  the  arteries  are  filled  with  one  color  and 
•the  veins  with  another  are  pretty  to  look  at,  but  are 
difficult  to  prepare,  and  present  no  practical  advan- 
tage. The  gelatine  solution  is  made  as  follows: 
Ten  grammes  of  clear  gelatine  are  cut  into  small 
pieces,  and  placed  in  a  beaker  of  cold  distilled  water 
to  soak.  In  about  an  hour  the  gelatine  will  have 
swollen  to  several  times  its  original  volume.  The 
excess  of  water  is  now  poured  off,  a  glass  cover  put 
over  the  beaker,  and  it  is  placed  in  a  water-bath  and 
heated  until  the  gelatine  is  rendered  fluid. 

For  the  red  injection  four  grammes  of  carmine  are 
rubbed  up  in  a  mortar  with  eight  cubic  centimetres 
of  liquor  ammonise,  and  then  fifty  cubic  centimetres 


MATERIAL    FOR    RED    INJECTION.  163 

of  water  are  added.  When  the  carmine  is  as  com- 
pletely as  possible  dissolved,  the  liquid  is  filtered. 
The  process  of  filtration  occupies  some  time — several 
hours,  in  fact — and  may  be  conveniently  left  over- 
night. The  filtrate  is  warmed,  and  the  gelatine  solu- 
tion is  gradually  added  to  it  with  constant  stirring. 
The  next  part  of  the  process  is  to  precipitate  the 
carmine,  for  otherwise  it  would  difl'use  through  the 
walls  of  the  vessels  and  color  the  tissues ;  but  it  must 
he  precipitated  so  finely  that  the  particles  shall  not 
be  visible  even  under  the  highest  power  of  the  micro- 
scope. To  effect  this  object  a  small  quantity  of  a  ten 
per  cent,  solution  of  acetic  acid  is  placed  in  a  burette 
and  allowed  to  run  drop  by  drop  into  the  warm  car- 
minized,  gelatine  solution,  which  is  all  the  while 
constantly  agitated.  As  its  alkalinity  becomes  neu- 
tralized the  amrnoniacal  odor  becomes  less  and  less 
strong,  and  eventually  disappears,  and  is  replaced  by 
the  vinegar-like  smell  of  acetic  acid.  The  alteration 
in  reaction  may  be  shown,  in  spite  of  the  red  color 
of  the  solution,  by  placing  a  small  drop  on  a  piece  of 
blue  litmus  paper ;  if  the  opposite  side  of  the  paper  be 
looked  at,  it  will  be  found  to  have  assumed  the 
characteristic  bright-red  color  which  acids  produce, 
and  which  is  quite  different  from  carmine.  This 
change  is  owing  to  the  diffusion  of  the  acetic  acid 
through  the  paper,  whereas  the  carminized  gelatine 
sets  almost  immediately  and  is  thus  unable  to  soak 
through. 

But  it  is  not  sufficient  in  order  to  effect  the  pre- 
cipitation of  the  carmine  that  the  fluid  should  only 
just  be  acidulated  ;  there  must  be  an  excess  of  acid. 
A  few  more  drops  are  therefore  added,  and  the  car- 
mine thrown  out  of  solution.  This  change  from 
the  soluble  to  the  insoluble  state  is  accompanied  by 
a  very  marked  alteration  in  color,  for  whereas  whilst 
still  in  solution  the  carmine  imparted  the  rich  deep 
red  of  an  ammoniacal  solution  to  the  gelatine,  after 
the  precipitation  the  color  of  the  latter  changes  to  a 
paler  red,  comparable  rather  to  the  tint  presented  by 


164  PRACTICAL    HISTOLOGY. 

the  powdered  carmine  in  the  dry  state.  Even  after 
the  production  of  this  change  a  few  more  drops  of 
the  acetic  acid  may  be  added,  for  it  will  do  no  harm, 
and  will  tend  to  counteract  the  natural  alkalinity  of 
the  tissues. 

The  colored  gelatine  is  next  filtered  through  a 
piece  of  flannel  or  fine  linen,  previously  soaked  in 
hot  water,  and  again  wrung  out,  and  is  collected  in 
a  flask  as  it  runs  through  the  filter,  and  transferred 
to  the  injecting  bottle. 

For  the  blue  injection  10  grammes  of  gelatine  are 
taken,  and  after  having  been  soaked  in  cold  water 
and  dissolved  up  as  before,  50  c.c.  of  a  2  per  cent, 
solution  of  Berlin  blue,  which  has  been  previously 
warmed,  is  gradually  added  with  constant  agitation 
to  the  fluid  gelatine.  The  blue  mixture  is  filtered 
and  is  then  ready  for  use,  without  the  necessity  of 
precipitating  the  coloring  matter,  for  ftiis  being  a 
colloid  is  indiiFusible. 

It  is  sometimes  advantageous  in  cases  where  the 
structure  of  the  walls  of  the  bloodvessels  is  to  be  the 
subject  of  observation,  to  use  an  injecting  mass  which 
is  far  less  deeply  colored.  This  can  of  course  be 
readily  obtained  by  diminishing  the  proportion  of 
carmine  or  Berlin  blue  which  is  used. 

The  soluble  Berlin  blue  is  of  great  value  for  the 
purpose  of  injecting  both  the  bloodvessels  and  lym- 
phatics. Unfortunately  it  is  very  troublesome  to 
prepare.  The  following  is  the  method  recommended' 
by  Briicke,  to  whom  we  owe  its  introduction : — 

Take  of  potassic  ferrocyanide  217  grammes,  and 
dissolve  in  a  litre  of  water  (solution  A). 

Take  a  litre  of  a  10  per  cent,  solution  of  ferric 
chloride  (solution  B). 

Take  four  litres  of  a  saturated  solution  of  sulphate 
of  soda  (solution  c). 

Add  A  and  B  each  to  two  litres  of  c.  Then  with 
constant  stirring  pour  the  ferric  chloride  mixture 
into  the  ferrocyanide.  Collect  the  precipitate  upon 
a  flannel  strainer,  returning  any  blue  fluid  which  at 


INJECTION    APPARATUS.  165 

first  escapes  through  the  pores  of  the  flannel ;  allow 
the  solutions  to  drain  off;  pour  a  little  distilled 
water  very  carefully  over  the  blue  mass,  returning 
the  first  washings  if  colored,  and  renew  the  water 
from  day  to  day  until  it  drips  through  permanently 
of  a  deep  blue  color.  This  is  a  sign  that  the  salts 
are  washed  away,  and  all  that  is  further  necessary 
is  to  collect  the  pasty  mass  from  the  strainer  and 
allow  it  to  dry. 

Apparatus  employed  for  injecting. — This  con- 
sists, in  the  first  place,  of  a  bottle  for  holding  the 
colored  fluid  ;  and  secondly,  of  some  means  of  pro- 
ducing a  steady,  elastic,  and  readily  alterable  pressure 
on  the  surface  of  the  fluid  so  that  it  may  be  driven 
with  any  required  force  into  the  arteries.  The 
method  formerly  employed  of  forcing  the  injecting 
material  from  a  small  syringe  directly  into  the 
bloodvessels  has  been  almost  entirely  given  up, 
on  account  of  the  impossibility  of  estimating  the 
amount  of  pressure  which  is  being  exerted,  leading 
often  to  the  employment  of  too  great  a  pressure  and 
the  consequent  rupture  of  some  of  the  smaller  blood- 
vessels. Fig.  28  represents  a  convenient  form  of 
apparatus  for  general  use.  The  bottle  (i),1  which 
holds  the  injecting  fluid,  is  a  moderate-sized,  wide- 
mouthed  phial,  with  a  well-fitting  vulcanized  India- 
rubber  cork,  through  which  two  glass  tubes  pass. 
One  of  these  goes  to  the  bottom,  and  from  it  an 
India-rubber  tube  passes,  which  will  be  subsequently 
connected  with  the  artery  canula  (c),  but  not  before 
this  has  been  inserted  in  the  bloodvessel,  in  the 
manner  immediately  to  be  described.  The  other 
passes  only  just  through  the  cork,  and  serves  to 
maintain  communication  by  means  of  another  India- 
rubber  tube,  with  the  pressure-bottle  p.  The  injec- 
tion-bottle is  placed  during  the  process  of  injecting 
in  a  large  beaker  (b)  of  warm  water  (about  40°  C.) ; 
a  piece  of  cork  is  wedged  in  between  the  bottle  and 

1  A  simil  .r  one  is  better  shown  in  Fig.  30,  at  c. 


166 


PRACTICAL    HISTOLOGY. 


the  side  of  the  beaker  to  prevent  the  bottle  from 
floating  up  as  it  becomes  emptied  of  injection,  and 


Fig.  28. 


Injecting  apparatus.     Complete. 

s,  condensing  syringe,  fixed  to  the  table  ;  p,  pressure-bottle  ;  6,  beaker  of  warm 
water  in  which  the  injection-bottle,  t,  stands  ;  ft',  small  beaker  containing 
salt  solution  ;  w,  water-bath  heated  by  a  ring  burner  below  ;  the  tempera- 
ture of  the  water  is  indicated  by  a  thermometer,  t,  placed  in  it ;  c,  arterial 
canula  connected  to  an  India-rubber  tube  from  the  injecting  bottle;  close 
to  the  canula  is  a  steel  clip.  The  canula  rests  upon  a  glass  plate,  which 
may  serve  either  to  put  the  animal  which  is  being  injected  upon,  or  to  cover 
it  over,  if  it  is  thought  necessary  to  place  it  in  the  water-bath. 

the  beaker  is  covered  with  a  glass  plate  (not  shown 
in  the  figure).     The  pressure-bottle  is  a  large  glass 


INJECTION    OF    BLOODVESSELS.  167 

or  earthenware  bottle  capable  of  holding  two  or 
three  gallons,  and  tightly  fitted  with  an  India- 
rubber  cork,  through  which  two  glass  tubes  pass. 
One  of  these  is  connected,  as  before  mentioned,  with 
the  injection  bottle,  and  the  other  with  a  condensing 
syringe  (s),  by  means  of  which  the  air  within  the 
bottle  can  be  brought  to  any  state  of  tension  that 
may  be  desired.  "Finally,  if  the  injection  is  to 
occupy  a  considerable  time,  a  water-bath  or  sand- 
bath  (?/?),  heated  by  a  ring-burner  to  about  40°  C., 
should  be  provided  for  receiving  both  the  beaker 
containing  the  injection-bottle  and  the  animal,  and 
maintaining  their  temperature  during  the  process. 
Ordinarily,  however,  if  the  operation  be  quickly 
and  dexterously  performed,  the  whole  process  will 
not  occupy  more  than  a  few  minutes,  and  will  be 
over  before  the  natural  heat  of  the  body  has  had 
time  to  become  dissipated. 

Everything  then  being  in  readiness,  the  animal,  a 
rabbit,  guinea-pig,  or  rat,  for  example,  is  killed  by 
chloroform  inhalation,  being  placed  under  a  bell- 
glass  with  a  sponge  wetted  with  chloroform.  The 
moment  it  has  ceased  to  breathe,  it  is  taken  out  and 
held  by  an  assistant,  whilst  the  operator  first  quickly 
reflects  the  skin  from  the  front  of  the  thorax  and 
then  makes  an  opening  in  that  cavity  just  over  the 
position  of  the  heart.  This  is  then  seized  near  the 
apex  with  blunt  forceps,  drawn  out  of  the  aperture, 
and  held  here  by  an  assistant,  The  aorta  is  then 
found,  the  point  of  a  pair  of  forceps  passed  under 
it  close  to  the  heart,  and  a  thread  ligature  drawn 
round  it.  A  snip  is  now  made  in  the  left  ventricle, 
and  an  arterial  canula  (Fig.  29,  e')  passed  through 
this  into  the  aorta,  in  which  it  is  tied  by  the  liga- 
ture. Then  by  means  of  a  pipette  a  little  warm 
water  or  salt  solution  is  passed  into  the  canula  so 
as  completely  to  fill  it  to  the  exclusion  of  air. 

^The  next  thing  to  do  is  to  connect  the  canula 
with  the  India-rubber  tube  which  brings  the  injec- 
tion from  the  bottle.  But  this  tube  must  first  be 


168  PRACTICAL    HISTOLOGY. 

completely  filled  by  the  injection,  so  that  it  contains 
no  bubble  of  air.  To  effect  this,  whilst  the  India- 
rubber  tube  is  kept  closed  by  the  strong  spring  clip 
with  which  it  is  provided,  the  air  in  the  apparatus 


Canulas  for  injecting.     Natural  size. 

c.1  c.2,  c.*,  glass  canulas  of  different  sizes  ;  c.4,  metal  canula :  it  is  sometimes 
more  easy  to  insert  than  the  glass  ones,  especially  into  fine  bloodvessels,  or 
into  lymphatics  ;  cl,  steel  clip  for  clamping  an  artery,  or  a  small  India-rubber 
tube;  a  and  b  are  intended  to  illustrate  the  mode  of  making  the  glass  ca- 
nulas ;  a,  glass  tube  which  has  been  heated  in  the  middle  in  the  blowpipe 
flame,  and  drawn  out  so  as  to  be  narrower  here  ;  b,  the  same  tube  after 
having  been  again  heated  (by  the  tip  of  the  flame),  and  drawn  out  at  the 
paints  a;  a;,  so  as  to  narrow  it  still  more  at  those  places  The  subsequent 
proceeding  consists  in  making  a  nick  at  I  with  the  edge  of  a  file,  breaking 
the  tube  across  here,  and  with  a  fine,  flat,  wetted  file  grinding  the  end  away 
obliquely  as  far  as  the  doited  ring  In  each.  The  sharpness  of  the  filed  edije 
is  got  rid  of  by  inserting  it  for  a  moment  or  two  in  (he  flame.  Two  similar 
canulas  are  thus  made  from  the  one  piece  of  tubing. 

is  put  under  a  pressure  of  about  two  inches  of  mer- 
cury by  working  the  syringe.  The  free  end  of  the 
India-rubber  tube  is  now  held  up,  and  the  clip 
opened  until  the  colored  fluid  forced  up  by  the  pres- 
sure begins  to  escape,  when  the  clip  is  immediately 
closed  and  the  tube  is  slipped  on  to  the  arterial 
canula.  The  greatest  care  must  be  taken  throughout 
to  avoid  the  introduction  of  air,  since  this  would 
obstruct  the  smaller  vessels  and  prove  fatal  to  the 
success  of  the  injection. 

The  clip  is  now  permanently  opened  and  the  injec- 
tion suffered  to  flow  into  the  aorta,  at  first  under 
the  low  pressure  of  two  inches  of  mercury  ;  but  the 


TREATMENT    OF    INJECTED    PARTS.         169 

pressure  is  gradually  increased  by  working  the 
syringe  until  a  pressure  of  four  or  five  inches  is 
attained.  The  blood  in  the  vessels  gets  forced  before 
the  injection  into  the  right  cavities  of  the  heart,  so 
that  these  are  soon  much  distended  ;  when  this  is 
the  case  the  right  ventricle  is  slit  open  and  the  accu- 
mulated blood  allowed  to  flow  out.  The  blood  is 
soon  followed  at  first  by  a  mixture  of  blood  and  the 
colored  gelatine,  but  afterwards 'by  the  latter  only  ; 
after  this  has  been  escaping  for  a  minute  or  two  the 
slit  in  the  ventricle  is  closed  by  placing  a  clip  on, 
or  tying  a  tape  round  the  heart,  and  the  injection 
being  thus  obstructed  in  its  outflow,  accumulates  in 
the  vascular  system,  and  distends  all  the  vessels  to 
their  fullest  extent.  The  success  of  the  injection 
may  be  estimated  by  the  color  of  those  parts  which 
are  not  concealed  by  the  fur ;  the  paws,  lips,  nose, 
and  ears,  for  instance,  and  the  tongue  and  interior  of 
the  mouth.  After  waiting  a  few  minutes  longer, 
until  the  vessels  may  be  considered  to  be  com- 
pletely filled,  a  tape  ligature  is  put  round  the  base 
of  the  heart,  so  as  to  include  all  the  great  blood- 
vessels, and  is  slowly  tightened.  This  will  effectu- 
ally prevent  any  escape  of  the  fluid  gelatine  from 
the  vessels  when  the  canula  is  removed  from  the 
aorta,  which  may  therefore  now  be  done,  the  clip 
having  been  first  replaced  on  the  India-rubber  tube 
which  is  connected  with  it,  so  as  to  prevent  the  in- 
jection from  spirting  out. 

The  animal  is  now  put  aside  until  it  has  become 
quite  cold  and  the  gelatine  is  firmly  set.  The  abdo- 
men is  then  freely  opened,  and  the  skull-cap  removed 
(if  the  brain  is  wanted),  and  incisions  having  been 
made  through  the  skin  of  the  limbs  here  and  there, 
the  animal  is  placed  overnight  in  weak  spirit  (equal 
parts  of  water  and  spirit),  and  the  next  morning 
those  parts  and  organs  which  their  color  shows  to 
be  well  injected  are  carefully  dissected  out  and 
placed  in  rather  stronger  spirit.  In  another  day  or 
two  they  may  be  transferred  to  the  strongest  spirit, 
15 


170  PRACTICAL    HISTOLOGY. 

in  which  they  will  soon  become  hard  enough  for  the 
preparation  of  sections.  The  object  of  trms  gradu- 
ally increasing  the  strength  of  the  spirit  is  to  prevent 
as  much  as  possible  the  shrinking  of  the  gelatine 
which  otherwise  results. 

The  blue  injection  possesses  the  material  disad- 
vantage that  it  is  apt  to  become  temporarily  reduced 
in  the  smaller  vessels  and  rendered  almost  entirely 
colorless,  so  that  it  is  difficult  to  determine  whether 
a  successful  injection  has  been  made  or  not.  The 
color  may,  however,  be  readily  restored  by  pouring 
some  oxidizing  fluid,  such  as  a  weak  solution  of 
peroxide  of  hydrogen,  over  any  part  about  which 
there  is  doubt ;  and  in  the  ordinary  course  of  pre- 
paring and  mounting  sections,  the  blue  color  is 
always  brought  back,  especially  if  turpentine  is  sub- 
stituted for  oil  of  cloves. 

Most  other  forms  of  apparatus  which  are  used  for 
injecting  are  more  or  less  like  the  one  above 
described,  the  chief  modification  being  in  the  mode 
in  which  the  pressure  is  produced,  this  being  effected 
in  one  form  by  allowing  water  to  flow  from  a  tap 
into  the  pressure-bottle  (which  in  such  cases  is  gene- 
rally made  of  metal),  and  thus  compressing  the  air ; 
in  another  by  allowing  mercury  to  flow  from  one 
vessel  into  another.  But  the  latter  method,  al- 
though useful  when  small  quantities  of  injecting 
fluid  only  are  required,  as  with  the  injection  of  the 
lymphatics  (with  which  the  apparatus  will  be 
described),  is  costly  for  large  quantities  ;  indeed  it 
will  be  found  that  none  are  more  simple  and  efficient 
in  working  than  the  one  here  recommended. 

If  a  condensing  syringe  is  not  at  hand,  sufficient 
pressure  may  be  got  in  many  cases  merely  by  blovring 
air  into  the  pressure-bottle  through  an  India-rubber 
tube,  its  escape  being  prevented  by  subsequently 
clipping  the  tube. 

Injections  which  are  fluid  in  the  cold  (of  which 
the  best  is  a  one  or  two  per  cent,  solution  of  Berlin 
blue)  are  sometimes  used  for  the  bloodvessels,  espe- 


TREATMENT    OF    INJECTED    PARTS.         171 

cially  for  injecting  cold-blooded  animals,  but  they 
do  not  as  a  rule  yield  such  good  results  as  a  success- 
ful gelatine  injection. 

Any  of  the  colored  gelatine  that  may  remain  over 
can  be  preserved  (even  for  a  considerable  time)  until 
again  wanted,  if  the  precaution  is  taken,  after  dis- 
connecting from  the  pressure-bottle  and  allowing  the 
fluid  in  the  canula  tube  to  ran  back,  to  place  the 
bottle,  tubes  and  all,  for  a  few- minutes  in  boiling 
water,  and  whilst  still  hot  to  stopper  up  the  ends  of 
the  tubes  with  pieces  of  glass  rod.  The  whole  can 
then  be  put  away  until  wanted ;  but  it  is  as  well  to 
heat  it  up  now  again  in  boiling  water,  to  destroy  any 
germs  of  fungi  which  may  perchance  have  entered 
the  bottle. 


172  PRACTICAL    HISTOLOGY. 


CHAPTER  IX. 

LYMPHATIC  AND  SEROUS  MEMBRANES. 

IT  is  in  preparations  of  the  serous  membranes  that 
the  structure  and  arrangement  of  the  lymphatic  ves- 
sels can  be  best  demonstrated,  and  it  will  on  this 
account  be  convenient  to  combine  them  here  under 
one  head,  especially  as  the  method  which  on  the 
whole  exhibits  the  structure  of  the  serous  mem- 
branes best  is  the  only  one  which  shows  at  all  satis- 
factorily the  structure  of  the  lymphatic  vessels  and 
their  relation  to  the  cell-spaces  of  the  connective 
tissue. 

Preparations  1,  2,  and  3.  Preparation  of  the 
rabbit's  omen  turn. — A  rabbit  having  been  killed 
by  bleeding,  the  abdomen  is  opened,  and  the  omen- 
turn,  which  is  generally  to  be  found  crumpled  up 
close  beneath  or  to  the  left  of  the  stomach,  is  raised 
with  forceps,  cut  off  as  close  to  the  line  of  attachment 
to  the  stomach  as  possible,  and  placed  in  a  shallow 
dish  of  salt  solution  which  is  at  hand  to  receive  it. 
Besides  this  salt  solution  there  should  be  ready  on 
the  table  a  little  one-half  per  cent,  solution  of  bichro- 
mate of  potash  in  a  bottle,  some  one-half  per  cent, 
solution  of  nitrate  of  silver,  a  wash-bottle  of  distilled 
water,  a  flat  glass  dish  containing  a  mixture  of  spirit 
and  water  (equal  parts),  two  glass  plates  about  four 
inches  by  six,  a  large  soft  camel-hair  brush,  and  two 
or  three  clean  capsules  and  watch  glasses. 

Two  small  corners  are  first  to  be  cut  off  the  omentum. 
One  of  these  is  placed  in  the  bichromate  of  potash, 
put  aside,  and  examined  after  two  or  three  days;  this 
is  for  exhibiting  the  arrangement  of  the  connective 
tissue  fibres.  The  other  is  first  rinsed  with  distilled 


PREPARATION    OF    THE    OMENTUM.          173 

water;  then  placed  for  one  minute  in  a  watchglass 
containing  a  little  of  the  silver  solution  ;  rinsed  again 
with  distilled  water,  and  exposed  to  the  sunlight  in 
another  watchglass  containing  water.  After  from  a 
few  minutes  to  half  an  hour  of  exposure,  according 
to  the  intensity  of  the  light,  it  may  be  removed,  and 
a  portion  or  the  whole  of  it  cautiously  mounted  by 
being  floated  upon  a  slide  under  water.  The  excess 
of  water  is  removed  from  the  .slide,  all  creases  and 
folds  are  carefully  got  rid  of  in  the  same  way  as  with 
the  frog's  mesentery  before  described,  and  finally  a 
drop  of  glycerine  is  added  and  the  coverglass  super- 
posed. This  preparation  is  for  the  purpose  of  show- 
ing the  epithelioid  layer  which  covers  each  surface  of 
the  membrane.  If  only  a  portion  were  mounted,  the 
rest  may  be  placed  for  a  few  minutes  in  weak  logwood 
before  mounting;  in  this  way  the  nuclei  of  the  cells 
may  be  brought  to  view. 

But  while  this  second  corner  was  being  exposed 
to  the  light  the  preparation  of  the  rest  of  the  omen- 
turn  can  be  proceeded  with. 

In  the  first  place,  it  is  floated  on  to  one  of  the  glass 
plates  and  removed  from  the  fluid,  and  then  by  draw- 
ing gently  first  at  one  place  and  then  at  another  the 
creases  and  folds  are  gradually  removed,  and  it  is  in 
this  way  spread  out  as  an  exquisitely  delicate  mem- 
brane which  may  be  made  to  cover  the  whole  upper 
surface  of  the  glass  plate,  and  may  be  extended  round 
its  edges  so  as  to  reach  the  lower  surface.  As  soon 
as  all  folds  are  in  this  way  removed  from  the  part 
which  covers  the  upper  surface  of  the  plate  the  second 
glass  plate  is  applied  to  the  under  surface  of  the  first, 
and  the  membrane,  or  at  any  rate  its  greater  part,  is 
thus  maintained  in  an  extended  state.  Next,  the 
surface  is  gently  brushed  all  over  with  the  camel-hair 
pencil  moistened  with  salt  solution;  this  is  for  the 
purpose  of  removing  the  epithelioid  layer  from  that 
surface,  and  enabling  the  silver  solution  more  rapidly 
to  penetrate.  The  brushing  is  not  absolutely  essen- 
tial, for  in  many  parts,  especially  those  in  which  the 

15* 


174  PRACTICAL    HISTOLOGY. 

lymphatics  and  bloodvessels  are  most  numerous,  the 
epithelioid  layer  is  deficient,  or  at  least  incomplete 
and  modified. 

The  salt  solution  is  now  thoroughly  washed  from 
the  membrane  with  distilled  water,  and  without 
delay  a  quantity  of  nitrate  of  silver  solution  is 
poured  on  it  and  allowed  to  run  over  every  part  of 
the  exposed  surface.  After  five  minutes  it  is  again 
washed  with  distilled  water,  and  the  glass  plates, 
with  the  membrane  of  course  still  upon  the  surface 
of  the  upper  one,  are  placed  in  the  weak  spirit  con- 
tained in  the  flat  glass  dish.  The  dish  is  then 
covered  and  placed  in  the  sunlight  until  the  silver, 
as  evidenced  by  the  change  in  color,  is  fully  reduced. 
Small  pieces,  each  about  an  inch  square,  are  then  cut . 
with  sharp  scissors  from  various  parts,  floated  on  to 
slides  with  the  browned  surface  uppermost,  and  are 
exposed  to  the  air  for  a  few  minutes  to  allow  most 
of  the  spirit  to  evaporate,  leaving  them  in  water ; 
to  this  a  drop  of  glycerine  is  added,  and  finally  the 
cover-glass  is  superposed.  Or  the  cover-glass  may  be 
put  on  first,  and  then  glycerine  placed  at  the  edge 
and  allowed  to  diffuse  in  underneath  the  cover.  The 
preparation  is  completed  by  cementing  the  covering- 
glass  in  the  usual  way. 

Both  before  and  after  the  treatment  with  nitrate 
of  silver  it  may  have  been  noticed  that  the  delicate 
membrane  is  studded  all  over  with  patches  of  thicker 
tissue,  some  quite  small  and  insular,  others  extend- 
ing over  a  considerable  area.  These  patches,  which 
are  characterized  by  an  accumulation  of  lymphoid 
cells  and  by  the  small  size  of  the  epithelioid  cells  of 
the  surface  (there  being,  in  fact,  in  many  cases,  a 
loss  of  distinction  between  them  and  the  other  cellu- 
lar elements  of  the  membrane),  are,  at  least  the 
larger  ones,  provided  with  numerous  bloodvessels, 
the  epithelioid  lining  of  which  is  often  very  well 
stained  by  the  silver;  and  these  are  always  accom- 
panied by  one  or  more  lymphatic  vessels,  with  walls 
formed  by  the  characteristic  wavy  outlined  cells. 


TENDON    OF    THE    DIAPHRAGM.  175 

Preparations  4  and  5,  The  central  tendon 
of  the  diaphragm  with  its  serous  coverings. — 

The  thoracic  and  ahdominal  surfaces  of  the  dia- 
phragm present  importantdifferences  in  the  arrange- 
ment of  the  numerous  lymphatic  vessels  which  are 
distributed  upon  them.  To  see  both  properly  it  will 
be  necessary  to  sacrifice  two  animals.  It  is  best  to 
use  rabbits,  since  their  central  tendon  is  larger  in 
proportion  than  that  of  most  otber  mammals. 

For  the  preparation  of  either  side  both  thoracic 
and  abdominal  cavities  must  be  freely  opened,  the 
ventral  attachment  of  the  diaphragm  being  left  in- 
tact, so  that  that  muscle  remains  stretched  out.  A 
double  ligature  is  to  be  put  on  the  inferior  vena  cava 
in  the  thorax,  and  the  vessel  cut  between  the  two 
threads;  this  is  to  prevent  the  blood  in  the  vessel 
from  getting  over  the  membrane.  The  pericardium 
is  now  cut  away  from  the  upper  surface  of  the  dia- 
phragm, and  the  suspensory  ligament  of  the  liver 
from  the  lower.  The  surface  which  it  is  wished  to 
silver  is  then  brushed  pretty  firmly  with  a  camel- 
hair  pencil  wetted  with  distilled  water,  after  which 
a  few  drops  of  nitrate  of  silver  solution  are  allowed 
to  flow  over  it,  or  are  applied  with  the  brush.  After 
five  minutes'  contact  the  silver  solution  is  washed 
off  by  a  stream  of  distilled  water,  and  the  central 
tendon,  including  also  some  of  the  muscular  fibres 
which  converge  to  it,  is  carefully  removed,  pinned 
out  upon  a  loaded  cork  or  cake  of  wax,  with  the 
silvered  surface  uppermost,  and  exposed  to  the  sun- 
light either  in  water  or  weak  spirit.  When  dis- 
tinctly browned  it  is  removed  from  the  window, 
and  pieces  from  different  parts  are  cut  out  and 
mounted  in  glycerine. 

In  addition  to  these  preparations — which  exhibit 
more  especially  the  lymphatics  and  cell-spaces  of 
the  serous  membrane,  and,  on  the  abdominal  side, 
the  lymphatic  clefts  between  the  tendon  bundles — it 
is  useful  to  make  another  silvered  preparation,  un- 
brushed,  of  the  peritoneal  suiface,  a  third  animal 


176  PRACTICAL    HISTOLOGY.     . 

being  sacrificed  for  the  purpose.  This  serves  to 
show  the  epithelioid  layer  of  the  serous  membrane, 
with  the  differences  in  character  of  its  cells  in  dif- 
ferent parts,  the  cells  being  much  smaller  over  the 
interfascicular  lymphatics  than  elsewhere.  Amongst 
the  smaller  cells,  moreover,  may  be  seen  here  and 
there  the  minute  darkly-stained  angular  patches 
known  as  pseudostomata,  which  are  probably  merely 
accumulations  of  intercellular  or  ground  substance; 
and  also,  but  more  rarely,  the  true  holes  or  stomata 
surrounded  by  a  ring  of  small  cells,  and  leading  into 
the  lymphatic  below. 

Preparation  6. — Such  stornata  or  orifices  leading 
from  the  serous  cavities  into  lymphatic  vessels  are 
met  with  occasionally  in  preparations  from  most  of 
the  serous  membranes  in  mammals.  But  they  are 
especially  numerous  and  well  seen  in  the  peritoneum 
of  the  frog. 

A  male  frog  should  be  killed  for  the  purpose,  and 
the  intestines  and  stomach  removed  so  as  to  expose 
the  back  of  the  abdomen,  but  without  cutting  the 
mesentery  too  near  the  spinal  column.  If  the  trunk 
of  the  animal  is  now  placed  in  a  dish  of  salt  solution, 
and  the  posterior  part  of  the  peritoneum  carefully 
examined  under  that  fluid,  it  will  be  found  that  it 
does  not  closely  cover  the  vertebral  column,  great 
vessels,  and  other  structures  which  are  found  at  the 
back  of  the  abdomen,  but  is  separated  from  them 
by  a  large  lymph-space,  divided  from  the  serous 
cavity  by  a  membrane.  This  is  covered  on  the  one 
side  by  the  epithelioid  cells  of  the  peritoneum  ;  on 
the  other  by  those  'of  the  lymphatic,  and  to  the 
unassisted  eye  appears  to  form  a  complete  septum. 
Under  the  microscope,  however,  it  is  seen  to  be 
studded  by  very  numerous  apertures,  which  can  be 
seen  if  the  membrane  is  removed  and  examined  with 
a  high  power  in  salt  solution  merely.  But  to  study 
their  arrangement  with  reference  to  the  epithelioid 
cells,  and  also  to  obtain  a  permanent  preparation, 
the  septum  is  to  be  stained  with  silver.  With  this 


INJECTION    OF    LYMPHATICS.  177 

object  the  whole,  or  a  portion  only,  is  dissected  off 
under  salt  solution  (it  may  be  convenient  to  remove 
with  it  the  elongated  kidneys  which  adhere  to  it 
behind  and  to  cut  these  away  only  after  the  staining 
is  completed) ;  it  is  then  rinsed  in  distilled  water  to 
remove  the  salt ;  placed  in  silver  solution  for  one 
minute;  again  rinsed,  and  exposed  in  water  to  the 
light.  After  the  metal  is  reduced  the  preparation 
is  floated  on  to  a  slide,  and  with  the  usual  precau- 
tions to  avoid  folds  and  creases,  finally  mounted  in 
glycerine.  It  is  desirable,  before  mounting,  to  stain 
the  tissue  with  logwood  in  the  way  recommended 
for  the  unbrushed  silvered  piece  of  the  rabbit's 
omentum,  but  this  is  not  absolutely  necessary,  and 
much  increases  the  risk  of  producing  folds  in  the 
membrane. 

For  studying  the  structure  of  the  larger  lymphatic 
vessels,  as,  for  example,  the  thoracic  duct,  precisely  the 
same  methods,  both  for  teased  preparations  and  for  sec- 
tions, are  employed  as  were  used  for  the  larger  blood- 
vessels. 

INJECTION  OF  LYMPHATICS. 

The  minute  lymphatics  of  a  part  may,  where 
numerous,  generally  be  readily  displayed  by  simply 
sticking  a  very  fine  canula  into  the  tissue,  and 
forcing  a  colored  fluid  through  this.  The  best 
apparatus  for  the  purpose  of  obtaining  the  requisite 
pressure  is  the  small  mercury  apparatus  shown  in  the 
accompanying  figure  (Fig.  30).  The  mercury  con- 
tained in  the  bottle  a  compresses  the  air  in  the  pres- 
sure-bottle 6,  according  to  the  height  a  is  raised 
above  6,  this  height  being  regulated  with  the  great- 
est nicety  by  the  screw  d.  The  bottle  c  containing 
the  injection  fluid  communicates  by  one  tube  with 
the  pressure-bottle,  and  by  another  (which  passes  to 
the  bottom)  with  the  injection  canula  f.  Gelatine 
is  not  used  for  injecting  the  lymphatics,  but  almost 
always  injections  which  are  fluid  iu  the  cold.  Berlin 


178 


PRACTICAL    HISTOLOGY. 


blue  solution  (2  per  cent.)  is  often  employed,  but  the 
best  fluid  for  the  purpose  is  a  solution  of  alkanet  in 


Fig.  30. 


Mercurial-pressure  apparatus  for  injecting  lymphatics. 

a,  reservoir-bottle  containing  mercury  ;  b,  pressure-bottle  into  which  this  tends 
to  flow;  (?,  injection  bottle  containing  solution  of  Berlin  blue,  connected 
with  the  pressure-bottle  by  one  India-rubber  tube,  with  the  canula /by  a 
second,  and  with  a  small  manometer  by  a  third;  d,  handle  of  screw,  by 
turning  which  the  stage  on  which  the  bottle  a  rests  is  raised  or  depressed, 
and  the  pressure  increased  or  diminished  in  b  ;  e,  screw  clip  (opened);  g, 
spring  clip  (closed). 

turpentine,  which  readily  flows  into  the  lymphatics. 
The  canula  can  be  made  from  a  piece  of  glass  tube 
drawn  out  to  a  capillary  point,  but  the  best  are  long 
perforated  steel  needles  like  those  supplied  with  the 
I'nivaz  syringes,  and  as  fine  as  it  is  possible  to  pro- 


INJECTION    OF    LYMPHATICS.  179 

cure   them  (Fig.  31).     The  India-rubber  tube  con- 
nected with  the  canula  is  closed  by  the  clip  g. 

Fig.  31." 


Very  fine  perforated  steel  needle  for  injecting  the  lymphatics  of  a  part. 

Preparation  7. — the  mode  of  injecting  the  lym- 
phatics of  a  tendon  may  be  here  described  as  an 
example,  especially  as  the  subject  was  omitted  when 
studying  the  minute  structure  of  tendon.  One  of  the 
best  tendinous  structures  to  choose  for  the  purpose  is 
the  fibrous  aponeurosis  covering  the  tendon  of  the 
triceps  extensor  femoris  of  the  dog.  Two  sets  of  lym- 
phatics are  here  met  with — one  in  the  substance  of 
the  tendon,  consisting  for  the  most  part  of  vessels  ar- 
ranged conformedly  with  the  direction  of  the  fibres 
and  connected  at  intervals  by  transverse  branches, 
so  as  to  form  elongated  and  oblong  meshes ;  and  a 
superficial  one  in  the  areolar  sheath  which  covers 
the  aponeurosis,  consisting  of  vessels  forming  a  close 
plexus  with  polygonal  rneshes.  The  latter  plexus 
should  first  be  attempted.  Both  tube  and  canula 
being  completely  filled  with  the  injecting  fluid  to 
the  exclusion  of  air-bubbles,  the  clip  g  is  closed,  and 
the  canula  is  inserted  obliquely  for  half  an  inch  or 
more  into  the  areolar  sheath,  care  being  taken  not 
to  mess  the  surface  of  the  tissue.  This  will  proba- 
bly be  effected  after  one  or  two  trials,  provided  the 
canula  is  sufficiently  fine  and  sharp.  The  clip  is 
then  removed,  and  by  turning  the  handle  d,  and 
thus  raising  the  bottle  a,  the  pressure  is  put  on  to 
about  an  inch  of  mercury,  as  indicated  by  the  gauge 
attached  to  the  injection-bottle.  If  the  insertion  of 
the  canula  have  been  fortunate,  the  blue  or  red 
fluid  will  almost  immediately  begin  to  pass  into  the 
lymphatic  plexus,  but  should  there  be  no  result  the 
pressure  may  be  gradually  raised  to  about  two  inches ; 
higher  than  this  it  is  not  as  a  rule  advantageous  to 


180  PRACTICAL    HISTOLOGY. 

go.  If  there  is  still  no  result  the  canula  may  be 
pushed  a  little  further  in  the  sheath,  and  perhaps 
moved  a  little  to  one  side  or  the  other  in  the  hope  of 
thus  rupturing  a  lymphatic  and  gaining  an  entrance 
into  the  plexus.  Should  these  and  other  devices 
which  experience  may  suggest  still  fail,  the  clip  must 
be  replaced  and  another  insertion  tried  elsewhere. 
It  very  frequently  happens  that  the  injection  which 
escapes  from  the  end  of  the  canula,  instead  of  pass- 
ing into  the  lymphatics,  forms  merely  a  bulla  of  ex- 
travasated  fluid  in  the  interstices  of  the  tissue.  This 
can  sometimes,  by  passing  the  handle  of  a  scalpel 
over  it  with  moderately  firm  pressure,  be  induced 
to  find  its  way  into  the  absorbent  vessels,  but  if  not 
the  canula  must  be  withdrawn  and  re-inserted  as 
before. 

For  the  lymphatics  in  the  fibrous  substance  of  the 
aponeurosis  the  canula  must  be  inserted  obliquely 
into  the  tendinous  tissue,  and  the  injection  forced 
in  with  the  same  precautions.  The  pressure  may,  if 
necessary,  be  raised  somewhat  higher,  for,  owing  to 
the  firmness  of  the  tissue,  there  is  less  liability  to 
the  occurrence  of  extravasations. 

For  displaying  these  injected  preparations  they 
may,  if  injected  with  Berlin  blue,  be  first  placed  in 
spirit  to  remove  all  water  and  precipitate  the  color- 
ing matter  in  the  vessels  (the  process  being  completed 
by  putting  the  injected  part  into  absolute  alcohol), 
after  which  the  preparation  may  be  placed  in  turpen- 
tine and  mounted  in  dammar  varnish.  Another 
method,  and  one  which  succeeds  very  well,  especially 
with  the  alkanet  injection,  is  to  stretch  the  injected 
aponeurosis  over  a  ring  of  cork  and  allow  it  slowly 
to  dry  by  exposure  to  the  air.  When  completely 
dry  the  injected  part  may  be  at  once  mounted  in 
dammar  or  in  glycerine.  By  this  mode  of  proceed- 
ing the  injection  is,  as  pointed  out  by  Bowditch, 
rendered  more  complete,  for  the  fluid  which  may 
have  been  extravasated  in  the  interstices  of  the  tissue 
is  apt  to  become  drawn  into  the  lymphatic  vessels 


INJECTION    OF    LYMPHATICS.  181 

to  supply  the  place  of  the  watery  fluid  which  becomes 
lost  by  evaporation. 

Although  in  many  cases  it  is  better  to  use  an  ap- 
paratus of  the  kind  above  described,  which  enables 
the  pressure  which  is  being  employed  to  be  exactly 
estimated,  yet  it  may  be  stated  that  with  a  little 
experience  the  lymphatic  vessels,  especially  those  in 
the  firmer  tissues  and  organs,  may  often  be  injected 
with  great  success  by  using  simply  an  ordinary 
Pravaz  subcutaneous  syringe  provided  with  a  line 
canula,  driving  the  injecting  fluid  into  the  tissue 
merely  by  the  pressure  of  the  finger.  It  is  true  that 
extravasations  are  very  apt  to  be  produced  opposite 
the  point  of  the  canula,  but  these  can  often  be  util- 
ized in  the  manner  before  mentioned  by  gently 
pressing  on  them  and  endeavoring  to  induce  the  in- 
jection to  pass  into  the  lymphatic  vessels. 

In  rare  cases  a  vein  is  pierced  by  the  canula,  and 
the  system  of  blood  capillaries  of  the  part  is  then 
apt  to  become  filled,  but  both  the  vessels  themselves, 
and  the  meshes  they  form,  are  much  smaller  than 
the  lymphatic  capillaries,  and  a  knowledge  of  their 
general  appearance  and  mode  of  arrangement  in  the 
particular  tissue  will  prevent  any  error  from  arising 
in  this  way. 

Preparation  8. — The  lymphatics  of  the  dia- 
phragm may  be  injected  with  Berlin  blue  during 
life.  A  young  rabbit  is  chosen,  and  enough  chloral 
hydrate  is  injected  under  its  skin  to  anaesthetize  it 
completely  (about  8  minims  of  a  50  per  cent,  solution 
will  suflice).  The  skin  of  the  belly  is  then  cut 
through  for  a  couple  of  inches  close  to  the  ensiform 
cartilage,  and  the  edges  are  held  aside  by  an  assistant, 
and  the  muscular  wall  having  been  pinched  up  a. 
blunt-pointed  canula  is  passed  obliquely  into  the 
cavity  of  the  peritoneum  at  its  upper  part,  due  care 
being  taken  to  avoid  the  liver  and  stomach.  About 
five  cubic  centimetres  of  a  saturated  solution  of 
Berlin  blue,  previously  warmed,  are  now  injected 
through  the  canula,  which  is  then  withdrawn  and 
16 


182  PRACTICAL    HISTOLOGY. 

the  animal  put  aside  in  a  warm  place.  After  four 
hours,  during  the  whole  of  which  time  it  remains 
under  the  influence  of  the  chloral,  it  is  killed  by 
bleeding.  The  abdomen  is  then  opened,  and  the 
viscera  having  been  drawn  aside,  the  under  surface 
of  the  diaphragm  is  exposed,  and  the  blue  which 
covers  it  is  washed  off  by  a  stream  of  water.  If  the 
experiment  has  been  successful,  it  will  be  found  that 
the  whole  network  of  lymphatics  of  the  central  ten- 
don is  filled  with  the  blue  fluid ;  for  this,  assisted 
by  the  constant  respiratory  movements  of  the  dia- 
phragm, has  passed  from  the  peritoneal  cavity 
directly  through  the  open  stomata  into  the  lymphatic 
vessels.  The  tendon  may  be  cut  out  and  placed  in 
alcohol,  and  eventually,  after  passing  through  tur- 
pentine, mounted  whole  in  Canada  balsam,  between 
two  glass  plates,  and  used  for  examination  with  a 
low  power  of  the  microscope. 

THE  SYNOVIAL  MEMBRANES. 

These  structures,  which  are  to  be  regarded  as  free 
surfaces  of  the  ordinary  areolar  tissue,  and  present 
no  essential  differences  in  structural  appearance  from 
this,  may  be  prepared  for  microscopical  examination 
by  the  same  methods.  The  preparations  which  are 
of  greatest  value  are  those  stained  with  nitrate  of 
silver. 

Preparations  9,  10,  11. — Since,  as  is  always  the 
case  with  the  silver  method,  parts  should  be  as  fresh 
as  possible,  and  since,  moreover,  it  is  convenient  to 
have  large  joints  to  work  with,  a  neat's  foot  should 
be  procured  from  the  butcher's  unless  a  freshly 
amputated  limb  is  available.  In  the  neat's  foot  all 
three  kinds  of  sy  no  vial  membranes  may  be  found 
and  prepared.  The  mode  of  silvering  the  synovial 
bursee  is  quite  simple,  and  need  not  here  be  detailed  ; 
the  preparation  of  the  vaginal  synovial  membranes 
was  described  under  Connective  Tissue  (p.  65),  this 
being  taken  as  typical  of  the  structure  of  that  tissue ; 


SYNOVIAL    MEMBRANES.  183 

and  the  preparation  of  the  synovial  surfaces  of  the 
joints  was  given  under  Articular  Cartilage  (p.  93). 
But  in  the  last-mentioned  place  nothing  was  said 
as  to  the  mode  of  demonstrating  the  synovial  mem- 
brane proper,  for  we  had  there  to  do  only  with  the 
cells  and  cell  spaces  of  the  cartilage,  and  the  transi- 
tions met  with  to  those  of  the  connective  tissue  of 
the  membrane.  The  appearance  presented  by  the 
membrane  itself  is  shown  in  surface  sections  made 
in  a  similar  way  but  from  the  inner  surface  of  the 
capsule  of  the  joint.  It  will  be  seen  that  in  the  ox 
the  cells,  or  rather  in  the  silvered  preparations  the 
white  cell-spaces,  form  a  close  irregular  network  by 
the  union  of  their  processes ;  in  fact,  so  completely 
have  the  cells  become  extended  into  branches  that 
it  is  difficult  in  many  cases  to  make  out  where  the 
body  of  the  cell  has  been.  In  the  human  synovial 
membranes  this  is  not  the  case ;  in  fact,  the  appear- 
ances are  quite  characteristic  of  ordinary  areolar 
tissue.  The  arrangement  of  the  corpuscles  into  epi- 
thelioid  patches  is  not  frequent,  but  there  is  no  con- 
tinuous epithelioid  covering,  as  in  the  serous  mem- 
branes. Moreover,  the  lymphatics,  which  were  so 
numerous  in  those,  are  not  to  be  seen  in  the  synovial 
membranes,  although  capillaries  are  present  and  in 
many  places  approach  close  to  the  surface. 

Preparation  12. — But  to  study  the  characteristic 
arrangement  of  the  bloodvessels  of  the  synovial 
membranes  and  to  show  the  drcuhis  articuli  vascu- 
losus,  a  preparation  must  be  made  from  one  of  the 
joints  of  a  limb  that  has  been  fully  injected.  Sur- 
face sections  are  then  made  of  the  transitional  region 
where  the  synovial  membrane  terminates  on  the 
cartilage,  and  including  also  a  part  of  the  membrane. 
They  are  mounted  in  the  usual  way  in  dammar 
varnish,  but  without  being  stained. 

Preparation  13. — Fnally  the  Haversian  fringes, 
with  their  secondary  processes,  may  be  prepared. 
They  may  be  examined  fresh  in  salt  solution,  and 
may  also  be  obtained  from  the  joint  which  was 


184  PRACTICAL    HISTOLOGY. 

stained  with  nitrate  of  silver.  To  find  them  it  is 
best  to  immerse  the  joint  in  fluid,  for  by  this  means 
they  are  floated  up,  and  may  then  be  snipped  oif 
and  mounted. 

THE  LYMPHATIC  GLANDS. 

Preparation  14, — These  are  chiefly  studied  by 
means  of  sections.  They  are  hardened  in  strong 
spirit,  into  which  they  are  put  immediately  after 
removal  from  the  animal ;  in  two  or  three  days  they 
are  sufficiently  firm  to  cut,  but  improve  if  left  longer. 
The  lymphatic  glands  of  the  dog  may  be  recom- 
mended for  demonstrating  the  structure  of  these 
organs,  especially  for  showing  the  lymph-sinuses 
and  the  cortical  nodules.  The  gland  is  split  after 
hardening  into  two  equal  parts  by  a  longitudinal 
cut  through  the  hilus,  and  one  of  these  halves  is 
embedded  in  wax-mass  in  the  usual  way  (pp.  143, 
144),  and  with  the  artificial  surface  near  one  end  of 
the  wax  cake.  The  sections,  which  must  be  very 
thin  indeed,  and  should  include  both  cortical  and 
medullary  substance,  are  transferred  from  the  spirit 
to  water,  and  thence  to  a  slide,  and  mounted  in 
glycerine,  without  staining.  If  the  lymph-paths 
appear  filled  up  with  lymph  corpuscles,  so  that  the 
retiform  tissue  which  traverses  them  is  not  well  seen, 
but  the  whole  section  appears  more  or  less  uniform 
in  structure,  these  corpuscles  may  often  be  in  great 
measure  removed  by  vigorously  shaking  up  the 
sections  with  water  in  a  test  tube,  or  by  gently 
brushing  them  under  spirit  with  a  soft  camel-hair 
pencil.  Unfortunately  both  these  methods  tend  to 
break  up  the  sections,  and  indeed  it  is  not  necessary 
to  employ  them,  if  the  sections  are  made  sufficiently 
thin. 

These  organs  are  amongst  the  most  difficult  to 
demonstrate  the  structure  of  satisfactorily,  so  that  it 
may  be  well  to  defer  their  preparation  until  some 
practice  has  been  obtained  in  making  sections  of 
other  parts. 


THE    SKIN.  185 


CHAPTER    X. 

THE  SKIN,  HAIRS,  AND  NAILS. 

Preparations  1-6.    Sections  of  the  skin.— 

Portions  should  be  selected  for  examination  from 
different  parts  of  the  body  ;  the  palms  of  the  fingers 
or  toes,  the  scalp,  and  a  piece  from  some  part  of  the 
general  surface  ;  e.  g.  the  extensor  surface  of  the  fore- 
arm. The  skin  of  the  scrotum  may  also  be  prepared, 
to  show  the  bundles  of  plain  muscular  tissue  in  the 
subcutaneous  tissue  or  dartos,  and  a  small  piece  of 
the  ala  of  the  nose,  for  the  sake  of  the  well-marked 
sebaceous  glands,  which  open  into  the  follicles  of  the 
minute  hairs  found  in  this  situation. 

The  following  method  of  hardening  the  tissue  can 
be  recommended :  A  small  piece  only  is  removed, 
being  obtained  with  as  little  of  the  subcutaneous 
tissue  as  possible  adhering  to  it.  At  the  same  time, 
if  it  is  desired  to  examine  the  larger  sweat-glands, 
this  tissue  must  not  be  removed  too  freely,  since 
those  bodies  extend  down  into  it.  It  will  be  found 
that  the  fresh  skin  has  a  tendency  to  curl  in  at  the 
edges  ;  this  should  be  prevented  by  pinning  the  piece 
out  on  a  piece  of  cork.  The  latter  is  then  inverted 
into  a  beaker  containing  a  mixture  of  spirit  and 
chromic  acid  (equal  parts  of  spirit  and  of  half  per 
cent,  solution  of  chromic  acid).  Here,  while  the 
chromic  acid  hardens  the  tissue  pretty  uniformly, 
the  spirit  tends  to  prevent  the  epidermis  from  break- 
ing away,  as  it  is  apt  to  do  when  placed  in  chromic 
acid  alone.  (Simple  hardening  in  spirit  also  answers 
in  many  cases  fairly  well.)  After  having  been  a 
fortnight  in  this  mixture,  the  piece  of  skin  is  trans- 
ferred to  strong  spirit,  and  after  two  or  three  days 


186  PRACTICAL    HISTOLOGY- 

in  this  is  ready  for  embedding  and  making  sections. 
The  piece  from  the  palm  of  the  finger  is  to  be  cut  in 
two  directions,  viz.  (1)  across  and  (2)  parallel  with 
the  ridges  formed  by  the  papillae.  It  will  be  easier 
to  cut  a  thin  section  if  the  razor  is  made  to  travel 
from  the  corium  towards  the  epidermis,  rather  than 
in  the  opposite  direction.  The  pieces  of  skin  which 
contain  hairs  should  be  so  embedded  as  to  carry  the 
plane  of  the  vertical  section  in  the  direction  of  in- 
clination of  the  hairs,  so  as  to  gain  a  view  of  the 
hair  follicles  along  their  whole  extent,  and  to  show 
the  erectores  pili  if  possible. 

The  sections  are  in  each  case  to  be  placed  in  log- 
wood solution,  and  when  sufficiently  stained  to  be 
washed  in  water  and  transferred  successively  to 
spirit,  oil  of  cloves,  and  dammar  in  the  usual  way. 

The  preparations  may  be  much  improved,  so  far 
as  the  exhibition  of  the  epidermis  is  concerned,  by 
being  placed  in  a  saturated  solution  of  picric  acid 
for  about  half  an  hour  before  being  stained  with  log- 
wood. In  this  way  the  horny  parts  acquire  a  bright 
yellow  color,  which  contrasts  strongly  with  the  violet 
staining  of  the  Malpighian  layer.  Owing  to  the  num- 
ber of  nuclei  which  become  stained  in  it,  this  part 
of  the  epidermis  is  much  darker  than  the  papillary 
part  of  the  corium  which  is  in  contact  with  it.  In 
the  papillae  of  the  skin  of  the  finger  the  tactile  cor- 
puscles may  be  sought  for.  They  are  generally 
situated  quite  near  the  apex  of  the  papilla,  and  to 
see  them  well  it  is  important  to  cut  the  papillae 
exactly  vertically,  so  as  to  include  their  length.  In 
sections  which  have  been  somewhat  obliquely,  and 
in  sections  cut  parallel  instead  of  vertical  to  the  sur- 
face, the  transversely  or  obliquely  cut  papillae  appear 
as  round  or  oval  islands  in  the  midst  of  the  deeper 
cells  of  the  epidermis.  The  dentated  appearance 
presented  by  most  of  the  cells  of  the  Malpighian 
layer  can  readily  be  seen  with  a  high  power  immer- 
sion objective, 

Of  the  sections  made  from  the  skin  of  the  finger 


THE    SKIN.  187 

one  or  two  of  the  thinnest  should  be  mounted  in 
glycerine  as  soon  as  cut,  without  staining,  merely 
placing  them  first  in  water,  to  remove  the  spirit. 
The  fibrous-looking  tactile  corpuscles  can  generally 
be  made  out  better  in  these  than  in  the  preparations 
which  have  been  mounted  in  dammar. 

Preparation  7. — To  show  the  arrangement  of  the 
bloodvessels,  sections  of  skin  from  a  limb  which  has 
been  minutely  injected  may  be.  made  and  mounted 
in  dammar  by  the  usual  process.  These  must  either 
be  left  entirely  unstained  or  the  staining  must  be 
very  slight  indeed.  These  sections  will  generally 
include  clusters  of  fat-cells,  with  their  vessels. 

Preparation  8. — The  following  method  will  serve  for 
the  demonstration  of  some  points  in  the  structure  of  the 
corium,  as  well  as  the  arrangement  of  the  bloodvessels.1 
One  of  the  limbs  of  an  animal,  preferably  of  a  dog,  is  in- 
jected with  a  solution  of  Berlin  blue,  the  injecting  canula 
being-  placed  in  the  principal  artery  of  the  leg,  and  a  band 
being  tightened  firmly  over  the  upper  part  of  the  limb 
after  the  injection  has  been  flowing  for  a  minute  or  two, 
so  as  to  compress  everything  except  that  vessel  and  pre- 
vent the  further  escape  of  the  injection  from  the  veins  of 
the  limb.  A  pressure  of  from  four  to  eight  inches  of  mer- 
cury is  then  maintained  for  several  hours.  In  this  way 
all  the  vessels  become  completely  distended  with  the  blue 
fluid,  the  watery  part  of  which  in  large  measure  exudes, 
so  as  to  render  all  the  tissues  cedematous.  If  colored 
gelatine  is  employed  the  limb  is  all  the  while  kept 
thoroughly  warm  by  placing  it  over  a  large  water-bath 
and  covering  it  with  a  glass  plate  or  bell-jar;  and  the 
gelatine  in  the  injecting-bottle  and  supply-tube  must  also 
be  kept  fluid  by  similar  means.  After  the  time  mentioned 
the  artery  may  be  tied  and  the  limb,  if  gelatine  has  been 
employed,  removed  to  a  cool  place.  When  it  is  considered 
that  the  gelatine  is  entirely  set,  a  piece  of  the  skin  is  cut 
out,  stretched  over  a  dialyzing  glass  and  placed  in  a 
beaker  of  digestive  fluid,  which  has  been  previously  pre- 
pared by  adding  a  few  drops  of  a  glycerine  extract  of  the 

1  W.  Stirling  in  "Journal  of  Anatomy  and  Physiology,"  vol. 
x.  pp.  185  and  465. 


188  PRACTICAL    HISTOLOGY. 

gastric  mucous  membrane  to  five  hundred  c.  c.  of  a  0.2  per 
cent,  solution  of  hydrochloric  acid.  It  is  then  maintained 
for  five  or  six  hours  at  a  temperature  of  38°  C.,  when  the 
piece  of  skin  is  removed  and  placed  in  water  for  twenty- 
four  hours.  Sections  may  then  be  cut  in  any  desired 
direction,  stained  with  logwood,  and  mounted  in  glyce- 
rine ;  and  although  not  obtainable  very  thin,  yet,  owing 
to  their  clearness  and  transparency,  the  arrangement  of 
the  bloodvessels,  and  of  the  little  muscles  attached  to  the 
hairs  can  be  traced  with  comparative  facility. 

Preparation  9.  Hairs. — To  examine  a  hair,  all 
that  is  necessary  is  to  place  it  on  a  slide  in  a  drop 
of  water,  cover  with  a  thin  glass,  and  examine  with 
a  moderately  high  magnifying  power.  By  careful 
focussing  the  cuticular  scales  can  often  be  made  out 
on  the  surface  and  at  the  edges  of  the  hair,  especially 
on  the  small  hairs  of  the  general  surface  of  the  body. 
The  medulla  is  often  absent  in  hair  of  the  head,  but 
may  generally  be  found  in  those  of  the  beard  and 
whiskers.  Many  of  the  black  particles  which  are 
seen  in  a  hair  by  reflected  light,  and  especially  in 
the  medulla,  are  merely  small  globules  of  air  in  the 
interstices  of  the  tissue.  That  this  is  so  may  be 
proved  by  cutting  off  the  light  which  comes  from 
the  mirror  of  the  microscope  and  viewing  the  object 
by  reflected  light,  only  a  moderate  power  being  used. 
The  black  particles,  if  really  due  to  the  presence  of 
air,  will  then  appear  silvery  white,  just  as  in  the 
parallel  case  of  the  air  which  fills  the  lacunas  in  a 
section  of  hard  bone. 

It  will  be  useful  to  compare  the  appearances  pre- 
sented by  human  hair  with  those  exhibited  by  the 
hairs  of  some  of  the  common  domestic  animals. 
These  are  many  of  them  characterized  by  the  regular 
arrangement  of  the  medulla  (this  is  nearly  always 
present  in  the  hairs  of  quadrupeds),  which  forms 
different  patterns  in  different  kinds  of  animals,  so 
that  the  species  to  which  the  hair  belong  may  often 
be  determined. 


HAIRS  —  NAILS.  189 

Preparation  10. — The  fibrous  part  of  a  hair  can 
be  broken  up  into  its  constituent  fibres  and  cells,  if 
it  be  first  steeped  for  a  time  in  strong  sulphuric 
acid. 

Preparation  11. — The  relative  proportion  of  the 
three  constituent  parts  of  a  hair  to  one  another  is 
best  shown  in  transverse  sections. 

To  obtain  sectio'ns  of  hairs,  the  simplest  plan  is  to 
tie  a  number  together  and  dip  the  bunch  into  a 
strong  gum,  and  when  this  has  thoroughly  soaked 
in  amongst  the  hairs  to  remove  the  bunch  and  either 
let  the  gum  dry  and  harden  by  exposure  to  the  air 
or  plunge  it  into  spirit  containing  a  little  water,  by 
which  in  a  few  hours  the  whole  mass  is  rendered 
hard.  Sections  are  then  made  with  a  sharp  scalpel, 
and  are  placed  on  a  slide  in  water  and  covered.  The 
water  dissolves  the  gum  away,  and  all  that  is  now 
necessary  in  order  to  make  the  preparation  complete 
is  to.  allow  a  drop  of  glycerine  to  diffuse  under  the 
cover-glass,  which  may  then  be  secured  as  usual. 

The  hair-follicles  and  roots  of  the  hairs  are  seen 
in  the  sections  of  skin. 

Preparation  12. — The  nails  are  studied  by  means 
of  vertical  sections  made  both  longitudinally  and 
transversely.  The  finger  (or  toe)  should  if  possible 
be  previously  injected,  and  the  nail  with  the  matrix 
and  surrounding  skin,  having  been  removed,  may 
be  hardened  either  in  spirit  or  by  the  spirit  and 
chromic  mixture.  When  ready  for  cutting,  the 
piece  should  first  be  bisected  longitudinally ;  and 
one  of  the  halves  having  been  embedded  in  a  wax 
and  oil  mixture,  with  rather  an  excess  of  wax,  so  as 
to  render  it  harder  than  usual,  one  or  two  longitu- 
dinal sections  (which  need  not  be  very  thin)  a'long 
the  whole  length,  to  show  the  general  relation  of  the 
nail  to  its  matrix  and  to  the  epidermis,  are  to  be 
made  with  a  sharp  strong  scalpel.  These  need  not 
be  stained  ;  they  may  be  mounted  either  in  glycerine 
or,  if  injected,  in  dammar;  they  are  intended  only 
for  examination  with  a  low  power.  The  other  piece 


190  PRACTICAL    HISTOLOGY. 

may  also  be  embedded  in  the  same  mixture,  but  in 
such  a  way  that  the  laminae,  which  in  the  matrix 
represent  the  papillae  of  the  skin,  are  cut  transversely. 
The  sections  must  be  as  thin  as  possible,  and  stained 
either  with  logwood  alone  or  with  picric  acid  and 
logwood  in  the  same  way  as  was  recommended  for 
the  sections  of  skin,  before  being  mounted  in  dam- 
mar. 

But,  owing  to  the  substance  of  the  nail  being  so 
much  harder  than  the  subjacent  matrix,  it  is  very 
difficult  to  get  both  parts  equally  thin.  They  can, 
however,  he  got  of  much  the  same  degree  of  hard- 
ness by  means  of  the  gum  method.  The  piece  to  be 
cut,  which  should  be  quite  small,  is  placed  in  syrupy 
solution  of  gum  and  left  over-night ;  it  is  then 
transferred  to  a  mixture  of  spirit  with  one-sixth  of 
its  volume  of  water.  After  a  few  hours  the  gum 
which  has  penetrated  into  the  substance  of  the  tissue 
will  be  hardened  throughout,  and  the  mass  can  be 
embedded  and  cut  in  the  desired  direction,  the  knife 
being  wetted  with  some  of  the  same  spirit- mixture. 
Strong  spirit  should  not  be  used,  since  the  gum  is 
entirely  dehydrated  by  this,  and  becomes  so  hard  as 
to  turn  the  edge  of  the  knife.  Instead  of  embed- 
ding the  piece  in  wax-mass  it  will  be  found  a  cleaner 
and  more  convenient  method  to  make  a  slit  in  a 
winebottle  cork,  insert  the  hardened  guru-impreg- 
nated tissue  in  the  proper  position  in  the  slit,  and 
maintain  it  in  place  whilst  cutting  the  sections 
by  the  pressure  of  the  finger  and  thumb.  The  sec- 
tions are  transferred  from  the  spirit  to  water,  which 
dissolves  out  the  gum ;  when  quite  free  from  this 
they  are  stained  and  mounted  as  before. 


It  will  be  well,  before  commencing  the  descrip- 
tion of  the  mode  of  preparing  the  several  viscera 
for  microscopical  examination,  to  revert  to  one  or 
two  preparations  which  were  purposely  deferred 


LYMPH    SPACES.  191 

until  the  mode  of  preparing  sections  and  of  injecting 
the  bloodvessels  had  been  explained. 

Injected  muscular  tissue  is  obtained  from  any 
injected  limb,  and  should  be  hardened  in  strong 
alcohol.  If  the  piece  is  not  large  enough  to  hold 
in  the  fingers,  it  must  be  embedded,  and  moderately 
thin  sections  made  both  longitudinally  and  trans- 
versely, and  mounted,  unstained,  in  dammar. 

Transverse  sections  of  a  nerve-trunk  may  be 
made  from  any  large  nerve  that  has  been  hardened 
in  picric  acid  (forty-eight  hours),  and  subsequently 
in  alcohol.  The  sections  are  to  be  stained  with 
picro-carmine  (twenty-four  hours),  and  may  be 
mounted  in  glycerine.  By  this  method  the  medul- 
lary sheath  of  the  nerves  and  the  elastic  tissue  of 
the  perineurium  and  epineurium  are  stained  yellow, 
the  connective  tissue  lamellae  and  bundles  pale  red, 
and  the  corpuscles  and  nuclei  and  the  axis  cylinders 
a  darker  red. 

Longitudinal  sections  of  a  nerve-trunk  from  an 
injected  limb  may  be  prepared  in  the  same  way  as 
those  of  injected  muscle. 

The  lymph  spaces  which  lie  between  the  lamel- 
lae of  the  perineurium  of  the  nerve,  and  extend  also 
amongst  the  fibres  within  the  funiculi,  can  very 
readily  be  injected  with  2  per  cent,  solution  of 
Berlin  blue  by  merely  sticking  a  very  fine  injecting 
canula  into  a  funiculus,  and  employing  moderate 
pressure.  The  injection  runs  along  the  course  of 
the  nerve  almost  as  freely  as  if  it  were  an  open  tube. 
A  piece  of  nerve  which  has  been  injected  in  this 
way  is  to  be  cut  out,  hardened  in  spirit,  and  trans- 
verse sections  prepared  and  mounted  in  dammar, 
without  staining,  to  show  the  course  taken  by  the 
injection.  At  the  entrance  of  the  nerve-roots  into 
the  spinal  canal  the  perineural  clefts  communicate 
with  the  sub-arachnoid  space,  so  that  the  nerves  can 
be  injected  by  merely  forcing  the  injecting  fluid  into 
this. 


192  PRACTICAL    HISTOLOGY. 

Longitudinal  sections  of  ganglia,  both  spinal 
and  sympathetic,  may  be  prepared  from  specimens 
that  have  been  hardened  either  in  picric  acid  and 
spirit  or  in  2  per  cent,  bichromate  of  ammonia 
solution  for  five  days,  then  in  weak  spirit  (half 
water)  for  twenty-four  hours,  and  then  in  strong 
spirit.  The  sections  are  to  be  stained  in  logwood, 
and  mounted  in  dammar. 

The  sections  of  developing  bone  (p.  108)  (instruc- 
tions for  embedding  which  by  the  cacao-butter  pro- 
cess, were  previously  given)  may  also  be  now  pre- 
pared. 


THE    HEART.  193 


CHAPTER    XI. 

THE  HEART. 

Preparation  1.    The  cardiac  pericardium.— 

The  pericardium  which  covers  the  surface  of  the 
heart  is  prepared  by  the  same  methods  as  other  serous 
membranes.  Of  these  the  only  one  which  need  here 
be  described  is  that  by  nitrate  of  silver.  This  is  as 
follows  :  In  an  animal  which  has  just  been  killed  the 
thorax  is  freely  opened,  and  the  pericardium  having 
been  torn  open,  the  base  of  the  heart  is  secured  by  a 
tape  ligature,  the  great  vessels  being  then  cut 
beyond  the  ligature,  and  the  organ  removed  without 
allowing  its  surface  to  be  smeared  with  blood.  A 
part  of  the  surface  is  now  brushed  firmly  with  a  soft 
camel-hair  brush  moistened  with  distilled  water, 
with  the  object  of  removing  the  superficial  layer  of 
epithelioid  cells.  The  heart  is  next  dipped  for  a 
moment  in  distilled  water,  and  then  nitrate  of  silver 
solution  is  poured  over  the  whole  surface,  and  al- 
lowed to  be  on  it  for  three  minutes,  after  which  the 
organ  is  rinsed  again  in  distilled  water,  and  finally 
placed  in  spirit,  in  the  sunlight.  AVhen  sufficiently 
browned  it  is  removed  from  the  window,  and  left 
for  some  hours  until  the  surface  is  hardened  by  the 
alcohol.  Surface  sections  are  then  made  of  both  un- 
brushed  and  brushed  parts,  and  after  soaking  in 
water  for  a  minute  or  two  are  mounted  separately 
in  glycerine.  The  unbrushed  specimens  will  show 
the  epithelioid  layer ;  the  brushed  ones  should 
exhibit  the  subjacent  connective  tissue,  with  its 
cell-spaces,  lymphatics,  bloodvessels,  and  nerves. 

Preparation  2.— The  muscular  substance  of 
the  heart  is  studied  in  teased  preparations  and  in 
17 


194  PRACTICAL    HISTOLOGY. 

sections.  For  the  teased  preparations  the  heart  of  a 
young  animal  should  be  chosen,  since  in  these  the 
fibres  separate  more  readily  into  their  constituent 
cells.  A  very  small  shred  is  placed  in  a  compara- 
tively large  quantity  of  TV  per  cent,  osmic  acid  for 
ten  days  or  a  fortnight ;  it  is  then  broken  up  in 
water  as  minutely  as  possible,  and  the  preparation 
covered  and  scanned  with  a  high  power.  Numerous 
little  fragments  of  varying  shapes  will  be  found 
scattered  over  the  preparation.  On  careful  exami- 
nation it  will  be  apparent  that  each  possesses  a 
nucleus,  which  can  be  made  more  conspicuous  by 
allowing  a  little  dilute  logwood  solution  to  run 
underneath  the  cover-glass.  These-  little  fragments 
of  the  cardiac  muscular  tissue,  which  have  the  char- 
acteristic indistinct  striation  of  that  substance,  are 
the  cells  which  by  their  union  end  to  end  form  the 
fibres. 

Preparation  3. — To  show  the  arrangement  of  the 
fibres,  and  the  interstitial  tissue  and  vessels,  a  piece 
of  the  muscular  substance  is  to  be  placed  in  strong 
spirit.  In  two  or  three  days  it  will  be  firm  enough 
to  cut.  Sections  are  to  be  made  both  parallel  with 
and  across  the  direction  of  the  fibres  ;  they  are  to  be 
stained  with  logwood  and  mounted  in  dammar. 

Preparation  4.  The  endocardium. — To  dis- 
play the  endocardium  the  silver  method  again  comes 
into  requisition.  That  part  of  the  lining  membrane 
covering  the  septal  wall  of  the  right  ventricle  is  the 
best  to  prepare,  on  account  of  its  relative  smoothness. 
The  right  ventricle  is  opened  in  a  fresh  heart,  and 
the  outer  wall  removed  entirely,  and  then  a  large 
piece  of  the  smoothest  part  of  the  exposed  surface  of 
the  septum  is  sliced  oft'  with  a  razor.  A  part  only 
of  the  endocardium  of  the  detached  piece  is  brushed, 
as  in  the  case  of  the  pericardium,  and  the  whole  is 
then  washed  and  treated  with  silver  solution. 
After  three  minutes  it  is  put  into  spirit  as  before, 
and  when  browned  and  hardened  surface  sections 
are  cut  and  mounted  in  glycerine. 


THE  LYMPHATICS  OF  THE  HEART.   195 

Preparation  5, — In  addition  to  these  silvered 
preparations,  the  endocardium  should  be  examined 
in  the  fresh  state.  This  is  done  by  dissecting  off  a 
piece  of  the  membrane  in  salt  solution  and  examin- 
ing it  both  with  and  without  the  addition  of  acetic 
acid.  Other  portions  may  be  teased  out  with  a  view 
to  the  demonstration  of  the  elastic  muscular  tissue. 
The  methods  for  making  and  preserving  these  pre- 
parations are  the  same  as  were  employed  for  showing 
the  structure  of  the  coats  of  the  bloodvessels,  to  the 
description  of  which  the  student  is  referred  (p.  140). 
It  may  be  noted  that  in  some  animals — the  sheep, 
for  instance — the  peculiar,  large,  cubical  or  oblong 
cells  which  form,  in  series,  the  fibres  of  Purkinje  will 
be  found  in  the  endocardium.  They  are  about  the 
size  of  fat- vesicles,  which  are  also  found  in  the  endo- 
cardium in  this  animal,  but  the  two  could  hardly  be 
confounded,  for  the  cells  forming  Purkinje's  fibres 
have  a  clear  or  slightly  granular  nucleated  central 
portion  which  does  not  strongly  refract  the  light,  and 
a  striated  circumference,  which  is  apparently  con- 
tinued into  that  of  the  neighboring  cells;  whereas 
the  fat-cells,  although  they  may  also  occur  in  rows, 
and  may  be  of  much  the  same  size  as  the  cells  in 
question,  present,  by  virtue  of  their  strong  refracting 
power  on  light,  a  totally  distinct  appearance. 

Preparation  6.  Lymphatic  system  of  the 
heart. — If  the  fine  canula  of  a  Pravaz  syringe  filled 
with  Berlin  blue  solution  is  stuck  into  the  muscular 
substance  of  the  fresh  heart  at  any  part,  and  the  fluid 
is  forced  out  at  the  point,  the  injection  will  pass 
freely  into  the  lymphatic  interstices  between  the  mus- 
cular fibres,  and  if  the  tube  is  inserted  near  the  outer 
or  inner  surface,  will  find  its  way  into  the  lymphatics 
of  the  pericardium  or  endocardium,  which  can  in 
this  way  be  readily  displayed. 


196  PRACTICAL    HISTOLOGY. 


CHAPTER    XII. 

THE  LUNGS. 

Preparations  1-2.    The  pulmonary  pleura.— 

The  serous  membrane  which  covers  the  surface  of 
the  lungs  as  well  as  that  which  lines  the  wall  of  the 
thorax  is  prepared  hy  the  silver  process.  For  the 
pulmonary  pleura,  the  lungs  of  a  small  animal  that 
has  just  been  killed  are  to  be  removed  entire,  and 
moderately  distended  with  air  through  the  windpipe, 
the  bronchi  being  then  tied  and  the  two  lungs  sepa- 
rated. One  is  rinsed  for  a  moment  in  distilled  water, 
and  a  little  nitrate  of  silver  solution  is  allowed  to 
flow  over  the  surface;  after  the  lapse  of  a  minute 
this  is  washed  off  again  with  distilled  water,  and 
the  organ  is  then  immersed  in  a  beaker  of  spirit  and 
exposed  to  the  light. 

The  surface  of  the  other  lung  is  to  be  firmly 
brushed  with  a  wet  camel-hair  pencil,  to  remove  the 
epithelioid  cells  of  the  surface  before  treating  it  with 
silver  solution.  This  may  be  suffered  to  remain  a 
minute  or  two  longer  in  contact  with  it  than  with 
the  other  lung;  in  other  respects  the  treatment  is 
similar.  Both  preparations  are  left  in  the  light  until 
they  appear  sufficiently  stained,  after  which  they  are 
to  be  placed  on  one  side  in  the  spirit  for  twenty-four 
hours.  They  will  then  be  sufficiently  hard  to  render 
it  possible  to  shave  off  a  thin  slice  from  the  surface. 
The  sections  so  made  are  to  be  placed  in  water,  and 
subsequently  mounted  in  glycerine,  with  the  outer 
surface  uppermost. 

Preparation  3.  The  costal  pleura  is  to  be  pre- 
pared in  situ  after  the  removal  of  the  lungs  and  heart. 
That  of  one  side  may  be  brushed,  the  other  not;  on 


THE    LUNGS.  197 

the  latter  the  silver  solution  is,  as  before,  to  be 
allowed  to  remain  a  shorter  time  than  on  the  brushed 
part,  where  the  fluid  has  to  penetrate  into  the 
lymphatic  vessels,  and  into  the  substance  of  the  tis- 
sue. The  whole  thorax  may  then  be  cut  off  from 
the  rest  of  the  trunk  and  exposed  under  water  to  the 
light ;  or,  if  it  be  too  large  to  do  this  conveniently, 
a  piece  only  of  the  thoracic  parietes  on  each  side  is 
to  be  removed  and  pinned  out  on  to  a  loaded  cork, 
which  is  then  placed  in  a  dish  of  water  in  the  sun- 
light. When  stained  pieces  of  the  membrane  must 
be  carefully  dissected  off,  without  pulling  upon  or 
injuring  the  tissue  in  any  way,  floated  upon  a  slide, 
the  excess  of  water  poured  off  or  soaked  up  with 
blotting-paper,  all  creases  removed  from  the  mem- 
brane, and  finally  the  cover-glass  superadded,  with 
a  drop  of  glycerine. 

Preparation  4.  The  lung  tissue. — The  struc- 
ture of  the  lungs  themselves  is  best  shown  by  means 
of  sections.  The  tissue  is  hardened  in  the  following 
way:— 

The  organs  having  been  removed  from  the  chest 
of  a  recently  killed  rabbit  or  cat,  care  being  taken 
not  to  scratch  their  surface  with  the  broken  ends  of 
the  ribs,  a  glass  canula  is  tied  into  the  end  of  the 
trachea  (or  into  either  bronchus).  The  canula  is 
then  connected  by  an  India-rubber  tube  with  an  in- 
jection bottle,  which  is  filled  with  a  weak  solution 
of  chromic  acid  (£  per  cent.).  By  blowing  air  into 
the  tube  this  solution  is  made  to  flow  into  the  lungs 
so  as  to  distend  them  moderately.  The  trachea  or 
bronchus  is  now  tied  up,  the  canula  removed,  and 
the  lungs  are  immersed  in  a  large  quantity  of  a  solu- 
tion of  chromic  acid  of  similar  strength.  After  two 
days  the  fluid  is  changed,  \  per  cent,  solution  being 
substituted,  and  the  organs  are  cut  into  pieces,  to 
enable  the  fresh  fluid  more  readily  to  penetrate. 
After  a  week  more  in  this  the  pieces  are  placed  first 
for  twenty-four  hours  in  weak  spirit  and  then  in 
strong  spirit. 

17* 


198  PRACTICAL    HISTOLOGY. 

The  bits,  though  small,  will  probably  be  large 
enough  to  hold  in  the  hand  and  cut  without  embed- 
ding. Sections  may  be  made  both  across  and  along 
the  course  of  the  bronchial  tubes,  stained  with  log- 
wood (it  will  be  found  that  the  sections  must  be  left 
for  a  considerable  time  in  logwood,  for  they  stain 
with  difficulty),  and,  after  going  through  the  usual 
processes,  mounted  in  dammer. 

Preparation  5. — But,  owing  to  its  spongy  nature, 
it  will  be  found  almost  impossible  to  cut  very  thin 
sections  unless  the  interstices  of  the  tissue  are  filled 
with  some  firm  material,  and  the  following  directions 
may  accordingly  be  observed,  if  thinner  sections  than 
can  be  obtained  in  the  ordinary  way  are  desired.  A 
small  piece  only  (not  larger  than  a  kidney-bean)  of 
the  lung  hardened  as  above  described  is  placed  for 
two  days  in  alcoholic  solution  of  logwrood  Kleinen- 
berg's.1  The  piece  of  .tissue  will  have  been  stained 
throughout  of  an  intense  dark  violet  color,  and  will 
look  almost  black.  On  removal  from  the  staining 
fluid  it  is  transferred  through  alcohol  to  oil  of  cloves. 
After  an  hour  in  this,  by  which  time  the  oil  will 
have  had  time  to  penetrate  its  whole  thickness,  it  is 
put  into  melted  cacao-butter,  which  is  kept  in  the 
fluid  condition  by  a  temperature  of  not  more  than 
42°  C.,  and  allowed  to  lie  in  this  for  four  hours. 
An  oblong  cake  of  the  cacao-butter  having  been  pre- 
viously made  by  pouring  some  of  the  melted  fat  into 
a  paper  mould,  a  little  pit  is  scooped  in  it  near  one 
end,  and  the  piece  of  tissue,  now  soaked  through 

1  Kleinenberg's  solution  is  made  in  the  following  way  (Foster 
and  Balfour) : — 

(1)  Make  a  saturated  solution  of  crystallized  calcium  chloride 
in  70  per  cent,  alcohol,  and  add  alum  to  saturation.  (2)  Make 
also  a  saturated  solution  of  alum  in  70  per  cent,  alcohol.  Add 
(1)  to  (2)  in  the  proportion  of  1  :  8.  To  the  mixture  add  a  few 
drops  of  a  saturated  solution  of  haimatoxylin  in  absolute  alcohol. 

This  solution  may  be  used  in  very  many  csiscs  for  staining  sec- 
tions, in  place  of  the  ordinary  watery  solution  of  logwood  alum. 
Jt  may,  if  required,  be  diluted  with  the  mixture  of  1  and  2.  The 
stained  sections  are  placed  at  once  in  strong  spirit. 


THE    LUNGS.  199 

and  through  with  the  cacao-butter,  is  placed  in  the 
pit  in  a  position  convenient  for  making  sections,  and 
the  pit  is  then  filled  with  the  melted  cacao-butter. 
This  as  it  hardens  adheres  firmly  to  and  sets  into 
one  piece  with  the  cake.  When  quite  hard — a  pro- 
cess which  takes  a  considerable  time — sections  of 
the  tissue  are  made  with  a  razor  wetted  with  spirit. 
Since  all  the  cavities  are  filled  by  and  the  tissue  is 
thoroughly  impregnated  with  the  cacao-butter,  the 
whole  cuts  like  a  homogeneous  piece  of  this  substance, 
and  sections  can  be  made  as  thin  as  desired.  As  they 
are  cut  they  are  placed  in  oil  of  cloves,  which  removes 
the  cacao-butter  in  a  few  minutes,  even  in  the  cold  ; 
but  in  the  winter  season  it  is  better  to  accelerate  the 
solution  by  slight  warmth.  The  sections  can  then 
be  mounted  in  dammar. 

These  stained  sections  of  lung  may  be  first  ex- 
amined with  a  moderate  power,  but  afterwards  a 
power  of  400  or  500  diameters  should  be  employed, 
in  order  to  see  the  details  of  structure ;  the  ciliated 
epithelium,  muscular  layer,  and  cartilaginous  plates 
of  the  bronchial  tubes,  with  the  mucous  glands, 
nerves,  lymphatics  (seen  in  section  as  mere  clefts), 
and  patches  of  lymphoid  tissue  in  their  walls ;  the 
branches  of  the  pulmonary  artery  accompanying 
them ;  the  mode  in  which  the  terminal  air-tubes 
dilate  into  the  infundibula ;  the  air-cells  or  alveoli, 
almost  covered  with  a  network  of  capillaries  which 
are  seen  also  on  the  septa  between  the  alveoli,  pro- 
jecting fii;st  into  one  and  then  into  the  other  of  two 
neighboring  air-cells.  Where  they  run  vertically  the 
capillaries  appear  in  optical  section  as  circular  spots, 
looking  not  unlike  nucleated  cells.  But  the  exces- 
sively delicate  epithelium  of  the  air-cells  cannot  be 
well  seen  in  these  preparations,  for  the  epithelium 
cells  remain  almost  unstained,  and  it  is  not  easy  to 
differentiate  their  nuclei  from  those  of  the  closely 
subjacent  capillaries. 

Preparation  6.  Epithelium  lining  the  air- 
cells. — In  order  to  demonstrate  the  epithelium  cells 


200  PRACTICAL    HISTOLOGY. 

I 

we  make  use  of  the  nitrate  of  silver,  but  the  mode 
of  proceeding  is  somewhat  different  from  that  ordi- 
narily employed.  A  gelatine  mixture  is  made  by 
taking  ten  grammes  of  gelatine,  and,  after  soaking 
it  in  cold  distilled  water,  melting  it,  and  adding  if 
necessary  more  warm  distilled  water  until  the  mix- 
ture measures  100  c.c.  A  decigramme  of  nitrate  of 
silver  is  dissolved  in  a  little  distilled  water  and 
added  to  the  gelatine,  and  the  mixture  is  transferred 
to  a  glass  syringe,  which  is  kept  warm  over  a  water- 
bath.  An  animal — preferably  a  young  one — having 
been  killed,  the  lungs  are  removed,  a  glass  canula 
with  a  small  piece  of  India-rubber  tube  attached  is 
tied  into  the  trachea,  and  then  the  point  of  the 
syringe  is  slipped  into  the  open  end  of  the  India- 
rubber  tube,  and  enough  of  the  gelatine  mixture 
injected  into  the  lungs  to  distend  them  pretty  com- 
pletely. The  trachea  is  now  tied  and  the  canula 
removed  from  it.  The  lungs  are  then  put  aside  into 
a  cold  place  until  the  gelatine  within  them  has  fully 
set,  when  sections,  which  should  be  as  thin  as  possi- 
ble, are  made  with  a  razor,  either  not  wetted  at  all 
or  with  distilled  water  only.  The  sections  so  ob- 
tained are  placed  on  a  slide  in  glycerine,  covered, 
and  exposed  to  the  light.  As  soon  as  they  seem 
sufficiently  stained  they  may  be  examined  with  as 
high  a  power  as  possible,  for  the  purpose  of  making 
out  the  silver  lines  between  the  epithelium  cells. 

Preparation  7.  Bloodvessels  of  the  lungs. — 
Lastly,  the  pulmonary  vessels  are  to  be  injected  and 
sections  made  of  the  injected  lung.  The  red  gela- 
tine injection  may  be  used  ;  this  and  everything  else 
is  to  be  got  ready  just  in  the  same  way  as  for  the 
injection  of  the  aortic  system,  but  a  syringe  filled 
with  melted  cacao-butter  must  be  connected  with 
the  trachea  in  the  same  way  as  for  the  gelatine  mix- 
ture in  the  last  paragraph  ;  and  moreover  the  arte- 
rial canula  is  of  course  to  be  passed  through  tlie 
right  ventricle  and  tied  into  the  pulmonary  artery, 
instead  of  into  the  aorta.  Immediately  the  gelatine 


LARYNX    AND    TRACHEA.  201 

injection  has  been  set  flowing  through  the  pulmo- 
nary vessels  the  lungs  are  pretty  fully  distended 
with  the  cacao-butter;  the  pressure  is  then  raised 
in  the  injecting  apparatus  to  about  four  inches  of 
mercury.  The  left  ventricle  is  first  slit,  to  let  the 
blood  out  of  the  pulmonary  system,  and  then 
clamped,  to  prevent  the  escape  of  the  injecting  fluid, 
which  is  allowed  to  pass  in  for  a  few  minutes  until 
it  is  thought  that  the  vessels  must  all  be  completely 
filled  ;  this  can  be  partly  made  out  from  the  color 
which  the  lungs  assume.  The  trachea  and  the  base 
of  the  heart  are  then  ligatured,  and  the  whole  is  left 
for  some  time  to  cool,  so  that  both  gelatine  and 
cacao-butter  are  fully  set.  The  lungs  are  then  cut 
out  and  placed  in  weak  spirit ;  after  a  day  they  are 
transferred  to  strong  spirit,  and  in  another  clay  or 
two  sections  may  be  made  (mostly  vertically  to  the 
surface  of  the  lung),  placed  in  warm  oil  of  cloves,  to 
dissolve  out  the  cacao-butter,  and  then  mounted  in 
dammar.  If  it  is  desired  to  stain  the  tissue  some- 
what, so  as  to  show  the  general  structure  of  the  lung 
as  well  as  the  arrangement  of  the  bloodvessels  in 
the  same  preparation,  this  can  be  done  by  placing 
the  sections,  after  the  cacao-butter  has  been  dissolved 
out  of  them,  first  in  absolute  alcohol,  to  get  rid  of 
the  oil  of  cloves,  and  then  for  a  few  minutes  in 
Kleinenberg's  logwood  until  they  are  sufticiently 
colored.  The  transference  through  absolute  alcohol 
and  oil  of  cloves  into  dammar  is  then  proceeded 
with,  as  in  the  case  of  other  sections. 

Preparation  8.  Larynx  and  trachea.— The 
trachea  and  larynx  are  hardened  in  £  per  cent, 
chromic  acid  (ten  days),  the  hardening  being  com- 
pleted by  spirit,  and  the  sections,  which  may  be 
longitudinal  of  the  cartilaginous  part  and  transverse 
of  the  posterior  membranous  part,  are  to  be  stained 
with  logwood  (they  will  require  a  considerable  time), 
and  mounted  in  dammar.  It  will,  of  course,  be 
necessary  to  embed  the  tissue,  and  it  will  be  found 
advantageous  in  cutting  to  pass  from  the  mucous 


202  PRACTICAL    HISTOLOGY. 

membrane  outwards,  instead  of  vice  versd.  But, 
owing  to  the  great  difference  in  hardness  between 
the  cartilaginous  rings  and  the  rest  of  the  tissue,  it 
is  difficult  to  get  a  complete  section  equally  thin 
throughout. 

Preparations  9-10.  Bloodvessels  and  lymph- 
atics of  trachea. — In  addition  to  these  sections  flat 
preparations  showing  the  bloodvessels,  and  others 
showing  the  lymphatics  of  the  mucous  membrane, 
may  be  made.  The  former  are  got  from  any  animal 
that  has  been  injected  entire,  the  mucous  membrane 
being  dissected  off  and  mounted  in  dammar.  The 
lymphatics  are  readily  filled  by  the  puncture  method 
by  sticking  the  point  of  the  injecting  canula  into 
the  mucous  membrane,  and  forcing  a  little  Berlin 
blue  or  alkanet-turpentine  in.  It  will  hardly  ever  fail 
in  finding  its  way  into  the  numerous  lymphatics  of 
the  mucous  membrane.  The  injected  portion  is  dis- 
sected off  and  mounted  in  dammar  varnish  or  in 
glycerine. 

Teased-out  bichromate  of  potash  preparations  to 
show  the  separated  epithelial  cells  have  already 
been  made  (p.  67). 

Preparations  11-13.  Ductless  glands  of  the 
larynx  and  trachea. — The  thyroid  and  thy m us 
are  studied  chiefly  by  means  of  sections,  for  facili- 
tating the  preparation  of  which  the  glands  are 
hardened  in  alcohol.  They  should  not  be  put 
entire  into  this,  but  either  cut  into  pieces  or  deep 
cuts  should  be  made  into  their  substance,  so  that 
the  preservative  fluid  may  penetrate  rapidly.  It  is 
well  to  place  them  at  first  in  weak  spirit  (half  water) 
for  twenty-four  hours,  and  then  to  transfer  them  to 
the  strongest  possible,  in  which  they  are  allowed  to 
remain  for  a  few  days  until  hard  enough  to  cut  thin 
sections  from.  Like  most  organs  which  have  been 
hardened  in  alcohol  alone,  they  stain  very  readily. 
The  so-called  "colloid"  which  is  met  with  in  the 
vesicles  of  the  thyroid  is  colored  by  the  logwood. 
Both  glands  may  be  advantageously  obtained  from 


LARYNX    AND    TRACHEA.  203 

the  body  of  a  newborn  cbild,  which  should  if  possi- 
ble be  injected,  so  that  the  mode  of  distribution  of 
the  bloodvessels,  especially  those  of  the  thynms,  may 
be  displayed. 

The  concentrated  corpuscles  jf  Hassall  which  are 
met  with  in  the  thynms  can  be  seen  in  sections  of 
that  organ,  but  may  also  be  studied  isolated  in  pre- 
parations of  the  fresh  part  teased-out  in  salt  solution. 


204  PRACTICAL    HISTOLOGY. 


CHAPTER    XIII. 

THE  MOUTH  AND  PHARYNX. 

Mucous  membrane  of  the  mouth. — Portions  of 
the  lining  membrane  of  the  mouth  are  best  prepared 
in  the  way  recommended  for  the  skin,  viz.  by  being 
pinned  out  upon  a  cork  and  immersed  for  two  or 
three  days  in  a  mixture  of  equal  parts  of  spirit  and 
half  per  cent,  chromic  acid  solution.  Sections  of  the 
cheek  or  lip  may  also  be  readily  prepared. 

THE  TEETH. 

Preparation  1.    Sections  of  hard  tooth. — No 

preparations  exhibit  the  structure  of  the  teeth  better 
than  these.  The  hard  tooth  is  ground  down  first  on 
one  side  and  then  on  the  other,  until  a  thin  section 
only  remains,  and  this  is  mounted  in  hard  Canada 
balsam  in  such  a  way  that  the  air  still  remains  in 
the  dentinal  tubules,  the  lacunae  of  the  cement,  the 
interglobular  spaces,  and  other  minute  cavities  that 
may  be  present.  The  preparation  is  similar  to  that 
of  bone,  but  presents  greater  difficulty.  Such  speci- 
mens may  advantageously  be  purchased,  for  their 
preparation  involves  the  expenditure  of  a  large 
amount  of  time  and  labor;  unless  the  use  of  a  lapi- 
dary's wheel  can  be  obtained,  when  the  process  is 
much  facilitated.  They  should  in  every  case  be 
studied  first  with  a  low  power,  and  afterwards  with 
a  high  power  objective. 

Preparation  2.  Sections  of  softened  tooth. 
— But,  in  addition  to  the  facts  which  the  hard  spe- 
cimens will  show,  various  others  may  be  made  out 


THE    TEETH.  205 

in  teeth  which  have  been  softened  by  immersion  in 
an  acid. 

The  acid  generally  used  when  the  structure  of  the 
(decalcified)  dentinal  substance  only  is  to  be  investi- 
gated is  the  hydrochloric.  A  10  per  cent,  solution 
may  be  employed,  and  the  tooth  is  steeped  in  this 
until  entirely  soft,  after  which  it  may  be  preserved 
in  spirit.  Sections  are  to  be  made  in  planes  both 
parallel  with  and  across  the  direction  of  the  dentinal 
tubes. 

Preparation  3.  Dentinal  sheaths. — To  show 
the  sheaths  which  line  the  dentinal  tubules,  a  piece 
of  such  softened  tooth  is  transferred  to  strong  hydro- 
chloric acid  (contained  in  a  watch-glass,  which  is 
covered  by  another,  inverted).  In  this  it  may  be 
left  for  about  an  hour,  after  which  time  all  that 
will  be  found  is  a  tenacious'  soft  mass  occupying  its 
place.  If  some  of  this  be  removed  with  a  small 
pointed  piece  of  wood,  placed  on  a  slide,  covered,  and 
examined,  it  will  be  found  to  consist  wholly  of  fine 
tubular  threads — the  dentinal  sheaths.  These,  being 
composed  of  a  substance  which  resists  the  solvent 
action  of  strong  hydrochloric  acid  longer  than  the 
other  animal  tissues,  remain  for  a  time  visible  after 
the  rest  of  the  dentinal  substance  has  disappeared. 

Preparation  4.  Soft  tissues  of  the  teeth.— 
But  to  study  the  soft  tissues — that  is  to  say,  the 
pulp  and  odontoblasts  with  the  processes  which 
these  send  into  the  dentinal  tubules — we  must,  as 
in  the  parallel  case  of  bone,  employ  a  reagent  which, 
whilst  softening  the  hard  parts,  at  the  same  time 
preserves  and  hardens  the  soft  parts.  Of  these,  picric 
acid  is  the  best  to  employ.  The  freshly- extracted 
tooth  is  placed  in  a  saturated  solution  of  the  acid, 
and  crystals  of  the  latter  are  from  time  to  time 
added  as  required ;  the  solution  being  stirred  as  fre- 
quently as  possible  with  a  glass  rod.  It  is  well  to 
break  the  tooth  first,  so  as  to  expose  the  pulp  cavity, 
if  this  can  be  done  without  disarranging  the  con- 
tents too  much.  When  softened  throughout — this 
18 


206  PRACTICAL    HISTOLOGY. 

can  be  tested  by  attempting  to  pass  a  fine  needle 
through  it — the  tooth  is  placed  in  spirit,  which 
should  be  changed  day  after  day  until  it  ceases  to 
become  much  colored  by  the  excess  of  picric  acid. 
All  that  remains  to  be  done  is  to  cut  the  tooth  in 
halves  vertically,  and  embedding  one  of  the  halves 
in  a  hard  wax-mass,  cut  thin  slices  from  the  artificial 
section,  stain  these  with  logwood,  and  mount  them 
in  glycerine. 

Preparation  5.  Study  of  the  teeth  "in  situ." 
— Still  more  instructive  preparations  are  obtained  by 
softening  a  portion  of  the  lower  jaw  with  the  teeth 
in  situ,  and  making  sections  through  the  whole  struc- 
ture. It  is  best  to  take  the  jaw  of  a  small  animal — 
a  rat,  for  instance.  The  flesh  having  been  cleared 
away,  the  softening  is  effected  with  picric  acid  in 
the  way  above  described ;  'and  then,  after  due  immer- 
sion in  spirit,  the  piece  is  imbedded,  cut,  stained  with 
logwood,  and  mounted  in  glycerine  as  before. 

Besides  showing  the  teeth  and  the  way  in  which 
they  are  inserted  into  the  lower  jaw,  the  structure 
of  this  bone  is  itself  well  demonstrated.  At  the 
lower  part  the  constantly  growing  incisor,  which 
extends  in  the  rat  below  the  molars  to  the  back  part 
of  the  jaw,  exhibits  the  large  elongated  odontoblasts 
of  a  developing  tooth,  with  their  well-marked  denti- 
nal  processes  (fibres  of  Lent),  which  in  some  parts 
project  like  harp-strings  across  a  small  space  which 
intervenes  between  the  cells  and  the  dentinal  sub- 
stance. It  will  be  remarked,  also,  that  in  these  teeth 
the  most  newly-formed  layer  of  dentine  becomes, 
especially  near  its  junction  with  the  older  parts,  very 
intensely  stained  by  the  logwood.  This  is  the  case 
with  all  teeth  which  are  still  in  process  of  develop- 
ment. Carmine  has  not  the  same  action. 

Preparation  6.  Development  of  the  teeth. — 
For  the  study  of  the  development  of  the  teeth  sec- 
tions are  made  of  the  jaws  of  embryos  and  young 
animals.  Perhaps  the  most  convenient  to  choose  are 
newborn  rats,  since  sections  of  their  jaws  exhibit  not 


THE    TONGUE.  207 

only  the  mode  of  development  of  the  teeth,  but  also 
that  of  the  hair,  the  bone  of  the  lower  jaw  (which 
ossifies  in  the  connective  tissue  around  Meckel's  car- 
tilage), the  tongue,  and  many  other  parts.  The  pre- 
paration is  as  follows:  The  foetuses  or  young  animals 
are  decapitated,  and  the  heads  dropped  into  a  large 
beaker  of  one-sixth  per  cent,  chromic  acid.  After  a 
week's  time,  during  which  the  liquid  is  now  and 
then  stirred,  they  are  transferred  to  weak  spirit,  and 
in  twenty -four  hours  to  strong  spirit.  After  being 
in  this  a  day  or  two  they  are  ready  for  cutting. 
Either  the  lower  jaw  is  removed  and  imbedded  sepa- 
rately, or  the  whole  head  is  placed  in  the  mould,  and 
both  jaws  are  cut  simultaneously.  The  sections  are 
to  be  stained,  some  with  logwood,  some  with  carmine 
solution  (made  by  dissolving  two  grammes  of  carmine 
in  a  few  drops  of  ammonia,  and  diluting  with  water 
to  one  hundred  cubic  centimetres).  The  stay  in 
chromic  acid  may  not  have  been  long  enough  to 
remove  all  the  earth  from  the  partly  developed  bones 
and  teeth,  but  what  still  remains  is  so  small  in 
amount  that  it  will  not  prevent  a  thin  section  being 
made.  The  earlier  stages  in  the  development  of  the 
teeth  may  be  perhaps  seen  in  the  molar  region ;  the 
later  stages  comprising  the  development  of  the  dental 
tissues,  especially  the  dentine  and  enamel,  may  be 
studied  in  the  much  more  advanced  incisors,  wrhich, 
as  just  pointed  out,  extend  backwards  in  these  ani- 
mals through  the  greater  part  of  the  length  of  the 
jaw. 

THE  TONGUE. 

Preparation  7. — Small  portions  of  this  organ 
from  different  parts  are  hardened  in  two  per  cent, 
bichromate  of  potash  (fourteen  days),  and  subse- 
quently in  spirit,  and  are  imbedded,  so  as  to  cut 
vertically  to  the  surface  of  the  mucous  membrane. 
The  sections  are  stained  with  logwood,  and  mounted 
in  dammar  varnish.  A  double  staining  with  picric 


208  PRACTICAL    HISTOLOGY, 

acid  and  logwood  may  also  be  employed  in  the  same 
way  as  with  the  sections  of  skin  (p.  186).  The  strati- 
fied epithelium,  the  papillae  of  the  mucous  membrane, 
and  the  arrangement  of  the  muscular  fibres  as  well 
as  the  mode  of  termination  of  the  superficial  mus- 
cular fibres  in  the  connective  tissue  of  the  mucous 
membrane,  may  be  studied  in  these  sections.  Some 
of  the  sections  include  mucous  glands,  which  differ 
in  appearance  according  to  their  condition  at  the 
time  of  death.  If  they  had  not  been  recently  stimu- 
lated by  the  ingestion  of  food  or  otherwise,  the  cells 
of  the  gland  will  still  be  filled  with  mucus,  as  shown 
by  their  swollen  appearance,  and  by  their  becoming 
strongly  stained  by  the  logwood;  whereas,  on  the 
contrary,  if  they  have  recently  discharged  their 
secretion,  the  cells  will  be  granular  and  almost  color- 
less, and  there  will  be  indications  of  mucus  in  the 
lumina  of  the  ducts. 

Besides  the  mucous  glands  others  may  be  seen  in 
the  neighborhood  of  the  papillae  vallatas,  which 
secrete  no  mucus,  and  consequently  do  not  present 
the  above  differences  of  staining.  Their  ducts,  if 
the  section  pass  in  the  direction  they  take,  will  be 
found  to  open  almost  without  exception  into  the 
fossae  of  the  circum  vail  ate  papillae,  or,  at  all  events, 
near  those  parts  in  which  taste-buds  have  been 
found.  The  taste-buds  themselves  may  possibly  be 
seen  in  the  epithelium  on  the  sides  of  the  papillae 
vallatae,  and  also  in  that  which  covers  the  mucous 
membrane- near  the  root  of  the  tongue  on  each  side. 
The  description  of  other  modes  of  preparing  them 
will,  however,  be  deferred  until  the  organs  of  special 
sense  are  treated  of. 

Preparation  8.  Vessels  of  the  tongue. — Sec- 
tions should  also  be  made  of  an  injected  tongue. 
These  will  show  not  only  the  numerous  vascular 
loops  in  the  more  obvious  papillae  of  the  mucous 
membrane,  corresponding  in  number  with  the  micro- 
scopic secondary  papillae,  but  also  the  arrangement 
of  the  vessels  in  the  muscular  substance  of  the  organ. 


THE    SALIVARY    GLANDS.  209 

The  injected  specimens  are  much  improved  by  slightly 
staining  them  with  logwood. 

Preparation  9.  Palate  and  tonsils.— The  soft 
palate  and  the  tonsils  may  be  hardened  in  the  same 
way  as  the  tongue,  or,  preferable,  by  immersion  for 
a  week  in  £  per  cent,  chromic  acid  solution,  and  sub- 
sequent placing  in  spirit.  The  sections  are  stained 
with  logwood  and  mounted  in  dammar. 

THE  SALIVARY  GLANDS. 

Preparation  10.— These  organs  are  prepared  by 
placing  small  pieces  of  them  as  soon  after  death  as 
possible  in  a  mixture  of  spirit  and  J  per  cent,  chromic 
acid  solution,  equal  parts  of  each.  After  two  or 
three  days  they  are  transferred  to  spirit,  and  in  a 
day  or  two  more  will  be  ready  to  cut.  They  may 
also  be  prepared  by  merely  being  placed  in  strong 
spirit  for  three  or  four  days.  The  embedding,  stain- 
ing with  logwood  and  mounting,  are  effected  in  the 
ordinary  manner,  the  chief  difficulty  being  met  with 
in  the  fact  that,  owing  to  the  loose  way  in  which 
the  lobules  are  held  together  by  the  intermediate 
connective  tissue,  the  sections  are  very  apt  to  become 
broken  up  by  the  agitation  which  ensues  from  the 
mixing  of  the  fluids,  particularly  when  they  are 
transferred  from  spirit  to  water  or  to  a  watery  solu- 
tion of  logwood.  An  alcoholic  logwood  (Kleinen- 
berg's)  may,  however,  be  used,  and  in  any  case  it  will 
be  found  that  the  small  pieces  exhibit  all  the  details 
of  structure  quite  as  well  as  larger  ones ;  thin  sec- 
tions which  have  thus  become  broken  up  need  not 
therefore  be  rejected. 

Preparation  11. — The  difference  in  the  structure  of 
the  salivary  glands  previous  and  subsequent  to  the  state 
of  secretory  activity  is  best  studied  in  the  submaxillary 
of  the  dog,  the  animal  being  killed  in  the  one  case  after 
some  hours'  fasting,  in  the  other  a  short  time  after  food. 
The  glands  are  hardened  and  sections  are  prepared  in 
the  manner  above  described,  and  the  differences  in  the 

18* 


210  PRACTICAL    HISTOLOGY. 

appearance  of  the  aveoli  in  the  charged  and  discharged 
conditions  respectively  of  the  glands  are  noted. 

Preparation  12. — Most  of  the  ordinary  hardening 
solutions  (alcohol  and  chromic  acid,  for  instance)  con- 
siderably alter  the  salivary  cells,  so  that  in  sections  of 
the  glands  the  cells  are  scarcely  ever  seen  of  their  normal 
form  and  appearance.  Osmic  acid  preserves  them  in  a 
more  natural  condition  than  most  reagents,  so  that,  to 
study  the  individual  elements,  a  very  small  portion  may 
be  placed  in  a  1  per  cent,  solution  of  this  reagent,  and 
after  forty-eight  hours  broken  up  finely  in  a  drop  of 
water  on  a  slide.  The  preparation  may  be  preserved  in 
glycerine. 


THE    (ESOPHAGUS    AND    STOMACH.  211 


CIIAPTEE    XIY. 

THE  (ESOPHAGUS  AND  STOMACH. 

Preparation  1. — The  oesophagus  is  best  hardened 
for  the  preparation  of  sections  by  a  mixture  of  equal 
parts  of  chromic  acid  (J  per  cent,  solution)  and  spirit. 
After  three  or  four  days  in  this  the  tissue  may  as  usual 
be  transferred  to  spirit.  Before  putting  it  into  the 
mixture  it  should,  if  but  a  small  piece  be  employed, 
be  pinned  out  upon  a  piece  of  cork,  so  as  to  stretch 
it  slightly  and  avoid  folds.  But  if  a  tubular  piece 
be  available  this  object  may  be  effected  more  satis- 
factorily loy  distending  the  organ  with  the  preserva- 
tive solution  through  a  glass  canula  tied  into  one 
end,  the  6ther  end  having  been  secured  by  a  ligature 
before  the  distension  ;  the  piece  is  then  immersed  in 
the  mixture  for  twenty-four  hours,  after  which  it 
may  be  cut  open.  In  embedding  the  gullet  and 
other  membranous  parts,  of  which  it  is  desired  to 
obtain  sections  vertical  to  the  surface,  it  is  well  to 
proceed  in  a  way  somewhat  different  from  usual,  the 
transfixion  with  a  pin  being  discarded.  A  layer  of 
the  melted  wax-mass  is  first  poured  into  the  mould 
so  as  to  fill  it  nearly  half-full  ;  the  piece  of  gullet  to 
be  embedded  is  deprived  of  all  superfluous  moisture 
by  placing  it  on  blotting-paper  for  a  few  seconds, 
and  then,  as  soon  as  the  wax-mass  in  the  mould  is 
hard  enough  to  support  its  weight,  it  is  placed  in  the 
desired  position  near  one  end  of  the  box,  which  is 
filled  up  with  more  wax-mass.  This,  especially  if  a 
few  degrees  above  its  melting-point,  adheres  firmly 
to  the  portion  of  the  wax-mass  which  was  first 
poured  in,  so  that  the  whole  forms  a  uniform  cake, 
with  the  tissue  embedded  in  it. 


212  PRACTICAL    HISTOLOGY. 

Moreover,  in  embedding  a  piece  of  one  of  the 
membranous  viscera  it  is  well  to  place  it,  as  a  general 
rule,  so  that  the  sections  shall  be  exactly  transverse 
to  the  axis  of  the  viscus,  following,  therefore,  the 
direction  of  the  circular  muscular  fibres  and  cutting 
the  longitudinal  across.  When  the  direction  of  the 
section  is  known,  it  is  easier  to  understand  the  ap- 
pearances wrhich  the  various  parts  present  when  cut. 
Sections  cut  parallel  to  the  axis  of  the  viscus,  and 
taking  therefore  the  direction  of  the  longitudinal 
muscular  fibres,  may  be  made  with  equal  advantage, 
but  oblique  directions  should  be  avoided. 

Preparation  2.  Bloodvessels  of  the  gullet— 
The  arrangement  of  the  vessels  is  best  shown  in  a 
flat  preparation.  A  small  piece,  obtained  from  an 
injected  animal,  is  transferred,  without  staining, 
from  the  spirit  to  oil  of  cloves,  being  subsequently 
mounted  in  dammar,  with  the  inner  surface  upper- 
most. Such  a  preparation  is  only  useful  for  exami- 
nation with  a  low  power,  but  by  this  the  arrangement 
of  the  vessels  in  the  successive  strata  can  be  well 
made  out. 

Preparation  3.  The  stomach.— The  stomach 
should  always  be  prepared  as  soon  as  possible  after 
death,  for,  in  the  first  place,  the  columnar  epithelial 
cells  covering  the  inner  surface  soon  become  altered  ; 
and  secondly,  if  digestion  were  proceeding  in  the 
organ  at  the  time  of  death,  the  mucous  membrane 
itself  becomes  attacked  by  the  gastric  juice  in  a  very 
short  time. 

The  abdomen,  therefore,  is  to  be  opened  as  soon 
as  the  animal  (a  cat  or  dog)  is  dead,  and  the  oesopha- 
gus cut  as  near  the  diaphragm  as  possible,  and  the 
duodenum  about  two  inches  beyond  the  pylorus  ; 
the  folds  of  peritoneum  connecting  the  viscus  to  the 
liver  and  neighboring  parts  are  also  severed,  and  the 
stomach  is  removed.  If  the  organ  is  empty  or  if 
the  contents  are  fluid  enough  to  admit  of  being 
poured  out  through  the  pylorus,  it  may  be  prepared 
as  a  whole  by  distension  with  the  spirit  and  chromic 


THE    GASTRIC    GLANDS.  213 

mixture  ;  spirit  alone  also  answers  very  well.  The 
duodenal  end  is  tied  up  and  a  glass  canula  is  fast- 
ened into  the  oesophageal  end.  This  is  connected 
by  an  India-rubber  tube  with  a  glass  tube  which 
passes  to  the  bottom  of  a  bottle  containing  the  "hard- 
ening fluid.  A  second  tube  passes  just  through  the 
cork  of  the  bottle,  and  by  blowing  through  it  the 
fluid  is  forced  into  the  stomach.  When  the  organ 
is  moderately  distended  the  India-rubber  tube  is 
clipped,  to  prevent  any  of  the  liquid  being  forced 
back  into  the  bottle  by  the  contraction  of  the  mus- 
cular walls  of  the  stomach  ;  the  gullet  is  then  secured 
by  a  ligature,  and  the  whole  organ  is  immersed  in  a 
large  bottle  or  covered  beaker  filled  with  the  same 
mixture  of  chromic  acid  and  spirit.  After  twenty- 
four  hours  it  should  be  opened  and  put  into  fresh 
fluid;  or,  if  it  is  not  desired  to  keep  the  whole  of 
the  organ,  small  pieces  only  from  different  parts  are 
so  transferred.  (Indeed,  if  the  stomach  be  too  large 
to  harden  as  a  whole,  or  difficult  to  be  cleared  of  its 
contents,  small  pieces  may  be  cut  out  from  the  fresh 
organ,  pinned  out  on  a  piece  of  cork  or  cake  of  wax, 
and  thus  immersed  in  the  spirit  and  chromic  mix- 
ture). In  two  or  three  days  more  the  tissue  is  hard 
enough,  if  the  spirit  be  of  the  strongest,  to  cut  sec- 
tions from,  and  small  pieces  may  accordingly  be 
embedded  with  this  view.  The  sections,  "which 
should  comprise  all  the  coats  of  the  organ,  are  to  be 
stained  with  logwood  and  mounted  in  dammar  as 
usual.  They  will  show  well  enough  the  relative 
thickness  of  the  several  layers  and  many  of  the 
structural  points,  but  for  making  out  distinctly  the 
structure  of  the  mucous  membrane  and  the  characters 
of  the  cells  which  occupy  the  gastric  glands  it  is 
necessary  to  make  thinner  sections  than  are  easily 
obtainable  in  conjunction  with  the  muscular  coat. 

Preparation  4.  Gastric  glands. — With  this 
object,  then,  small  pieces  of  the  fresh  mucous  mem- 
brane are  taken  from  two  distinct  parts,  one  from 
near  the  pylorus  and  the  other  from  the  cardiac 


214  PRACTICAL    HISTOLOGY. 

fundus,  and  placed  at  once  in  absolute  alcohol. 
When  hardened  they  are  embedded  separately,  and 
vertical  sections,  as  thin  as  possible,  are  made. 
Those  from  the  pyloric  piece  are  to  be  stained  with 
logwood  and  mounted  as  before  in  dammar,  but  one 
or  two  of  the  thinnest  may  be  selected  and  mounted 
in  glycerine.  Those  from  the  cardiac  piece  are  to  be 
stained  and  prepared  in  three  different  ways.  In 
the  first  place,  two  or  three  may  be  treated  as  just 
recommended  for  those  from  the  pyloric  piece. 
Secondly,  other  two  or  three  are  to  be  placed  for 
twenty-four  hours  in  a  weak  solution  of  carmine 
(the  carmine  solution,  p.  207,  diluted  five  times). 
Thirdly,  one  or  two  others  are  to  be  stained  with 
aniline  blue.  There  are  different  kinds  of  this  sold 
in  the  shops  ;  that  known  as  Nicholson's  No.  1  gives 
good  results.  A  one  per  cent,  solution  is  used,  and 
the  sections  are  left  in  for  about  thirty  minutes. 
They  are  then  placed  in  a  watchglass  containing  a 
mixture  of  glycerine  and  water,  equal  parts,  and  are 
finally  mounted  in  glycerine.  The  carmine-stained 
sections  are  also  mounted  in  glycerine,  after  having 
been  rinsed  in  water. 

These  different  modes  of  staining  bring  out  dis- 
tinctly the  differences  between  the  various  kinds  of 
cells  found  in  the  peptic  glands.  In  the  logwood 
preparations  the  peptic  cells  will  be  found  stained 
rather  less  than  the  rest,  whereas  in  the  sections 
stained  by  carmine  and  aniline  they  are  colored 
more  deeply.  This  is  especially  the  case  in  the  ani- 
line preparations,  where  the  peptic  cells  are  stained 
of  a  deep  blue,  whilst  the  other  cells  remain  almost 
colorless. 

Preparation  5.  Horizontal  sections. — Besides 
the  vertical  sections  of  the  mucous  membrane  others 
are  to  be  made  parallel  to  the  inner  surface,  and 
therefore  so  as  to  cut  the  glands  across.  To  effect 
this  a  small  piece  of  each  region  is  embedded,  with 
the  part  which  corresponds  to  the  inner  surface  of 
the  stomach  placed  opposite  the  end  of  the  mould. 


BLOODVESSELS    OF    THE    STOMACH.         215 

It  is  then  gradually  cut  into  slices,  which  will,  of 
course,  comprehend  in  succession  first  the  mouths  of 
the  glands,  then  their  necks,  and  finally  the  deeper 
parts.  The  sections  so  obtained  are  to  be  stained 
and  mounted  in  the  same  way  as  the  vertical  sec- 
tions. 

Preparation  6.  Cells  of  the  glands,  iso- 
lated.— In  addition  to  studying  them  in  sections 
in  this  way  the  cells  of  the  glands  are  also  to  be 
studied  in  teased-out  preparations  of  the  fresh  mu- 
cous membrane.  If  prepared  in  serum  they  will 
showT  better  than  by  any  other  method  the  characters 
of  the  different  kinds  of  cells.  The  cells  will  be 
more  readily  obtained  separate  if  a  small  piece  of 
the  membrane  is  placed  in  J  per  cent,  bichromate  of 
potash  solution  for  twenty -four  or  forty-eight  hours, 
but  the  cells  are  apt  to  be  somewhat  altered,  and  the 
columnar  cells  of  the  general  surface  and  mouths  of 
the  glands  to  become  transformed,  by  the  swelling 
and  escape  of  their  contained  mucus,  into  goblet- 
cells. 

Preparation  7.  Bloodvessels  of  the  stomach. 
— But  vertical  and  horizontal  sections  of  an  injected 
stomach  are  to  be  made.  This  may  be  obtained 
from  the  animal  which  was  injected  entire:  If  it 
were  a  rat,  the  preparations  are  to  be  made  from  the 
pyloric  half  of  the  organ,  since  in  this  animal  the 
cardiac  part  has  a  non-glandular  mucous  membrane 
with  stratified  epithelium  like  that  of  the  gullet. 
The  vertical  sections  need  not  be  very  thin ;  they 
are  improved  by  being  placed  for  a  few  minutes  in 
dilute  logwood,  so  as  to  become  slightly  colored, 
before  being  mounted  in  dammar  by  the  ordinary 
process.  Instead  of  cutting  horizontal  sections  a 
small  piece  of  the  injected  stomach  may,  if  from  a 
small  animal,  be  simply  mounted  flat  with  the  inner 
surface  uppermost,  without  staining. 

Preparation  8. — An  attempt  may  be  made  to  inject 
the  lymphatics  of  the  gastric  mucous  membrane  with 


216  PRACTICAL    HISTOLOGY. 

Berlin  blue,  but  the  process  requires  considerable  care 
and  experience,  since  it  presents  unusual  difficulties. 
Indeed,  it  is  only  quite  recently  that  attempts  to  fill  any 
lymphatics  except  those  in  the  deepest  part  of  the  mem- 
brane have  been  successful.  If  a  successful  result  is 
obtained  the  injected  portions  are  hardened  in  alcohol, 
and  vertical  sections,  which  may  be  tolerably  thick,  made 
and  mounted  in  dammar. 


THE    INTESTINE.  217 


CHAPTER    XV. 

THE  SMALL  AND  LARGE  INTESTINE. 

Preparation  1.    Sections  of  small  intestine. 

— Pieces  of  the  small  intestine  are  to  be  prepared  in 
exactly  the  same  way  as  the  stomach,  the  mixture 
of  alcohol  and  chromic  acid  solution  being  employed 
to  distend  the  gut,  which  is  then  immersed  in  the 
fluid.  After  a  few  hours  the  intestine  is  opened  arid 
the  fluid  changed,  and  in  three  or  four  days  the  tissue 
is  transferred  to  spirit,  to  complete  the  hardening. 
Three  pieces  of  the  small  intestine  are  to  be  pre- 
served in  this  way,  viz.,  one  from  the  very  com- 
mencement of  the  duodenum  (this  will  probably  have 
been  included  in  the  stomach  preparation) ;  a  second 
from  the  jejunum  ;  and  the  third  from  the  ileum,  in- 
cluding one  of  the  patches  of  Peyer.  The  pieces 
may  be  obtained  from  a  cat,  dog,  or  rabbit,  the  con- 
tents of  the  intestine  being  first  washed  out  by  forc- 
ing a  rapid  stream  of  the  alcohol  and  chromic  fluid 
through  them  before  tying  up  the  further  end. 
Instead  of  distending  it  with  the  preservative  fluid, 
the  gut  may  be  opened  and  kept  in  an  extended 
state  by  pinning  it  on  a  cork  or  cake  of  wax,  which 
is  then  inverted  into  the  fluid. 

In  embedding  the  small  intestine  in  wax- mass 
care  should  be  taken  that  the  inner  surface  does  not 
retain  too  much  spirit  between  the  villi,  for  this 
would  prevent  the  melted  wax-mass  from  penetrat- 
ing between  them,  so  that  they  are  thus  left  without 
a  support  whilst  being  cut.  At  the  same  time  the 
surface  should  on  no  account  be  allowed  to  become 
quite  dry. 

It  is  necessary,  in  order  to  see  the  structure  of  the 
19 


218  PRACTICAL    HISTOLOGY. 

villi,  that  the  sections  should  be  very  thin  indeed — 
so  thin,  in  fact,  as  to  include  not  the  whole  thick- 
ness of  a  villus,  but  only  a  longitudinal  slice ;  other- 
wise the  epithelium  on  its  surfaces  interferes  with 
the  view  of  the  internal  structure.  With  a  very 
sharp  razor  and  considerable  dexterity,  this  may  be 
effected  even  when  the  intestine  is  embedded  by  the 
ordinary  process,  but  the  thin  sections  so  obtained 
are  apt  to  become  broken  up  during  the  process  of 
staining  and  subsequent  washing.  It  is  therefore 
well  to  adopt  for  this  tissue  the  cacao-butter  process, 
described  at  p.  198.  A  small  piece  is  stained  with 
Kleinenberg's  logwood,  as  there  recommended,  and 
after  passing  through  alcohol  and  oil  of  cloves  is 
impregnated  with  melted  cacao-butter,  and  em- 
bedded in  a  cake  of  the  same  material.  There  is 
hardly  any  limit  to  the  thinness  with  which  sections 
from  a  piece  so  embedded  may  be  obtained,  and  all 
that  is  further  necessary  is  to  dissolve  away  the  in- 
cluded cacao-butter  from  the  sections  with  oil  of 
cloves  and  to  mount  them  in  dammar. 

Preparation  2.  Fat  absorption. — For  the  pur- 
pose of  studying  the  course  which  fatty  particles  take  in 
passing  from  the  cavity  of  the  intestine  into  the  central 
lacteals  of  the  villi,  an  animal  is  killed  three  or  four 
hours  after  a  meal  composed  almost  exclusively  of  fat  (it 
should  previously  have  been  allowed  to  fast  for  several 
hours).  On  opening  the  abdomen  the  lacteals  in  the 
mesentery  will  be  found  filled  with  chyle,  and  the  cavity 
of  the  small  intestine  occupied  by  emulsified  fat  which  is 
undergoing  absorption.  The  intestine  is  opened  at  once, 
and  two  or  three  very  small  pieces  of  the  mucous  mem- 
brane are  snipped  off  and  placed  in  1  per  cent,  osmic  acid 
solution  Another  minute  piece  is  placed  in  a  drop  of 
serum  or  aqueous  humor  and  is  quickly  teased-out  with 
needles ;  a  piece  of  hair  is  added,  and  the  preparation  is 
covered  and  examined.  One  of  the  portions  in  osmic 
acid  is  allowed  to  remain  forty-eight  hours  in  the  solu- 
tion, and  is  then  broken  up  in  water.  The  others  are 
transferred  to  dilute  Kleinenberg's  solution,  and  when 
stained  throughout  are  embedded  by  the  cacao-butter 


NERVES    OF    INTESTINE.  219 

process.  The  sections  are  placed,  after  the  cacao-butter 
has  been  extracted  from  them  by  warm  oil  of  cloves,  first 
in  spirit,  and  then  in  water,  and  are  finally  mounted  in 
glycerine. 

In  the  two  teased  preparations — serum  and  osmic — 
many  of  the  columnar  epithelium  cells  will  be  founi  to 
contain  fatty  globules  of  various  sizes  (stained  blaok  in 
the  osmic  preparation).  Similar,  but  for  the  most  part 
smaller  particles  will  also  be  found  in  the  numerous 
lymphoid  corpuscles  which  are  set  free  from  the  retiform 
tissue  of  the  mucous  membrane  by  the  process  of  teasing. 
In  the  sections  the  epithelium  cells  and  the  lymph  cor- 
puscles will  be  observed,  in  situ,  in  the  same  condition, 
viz.,  containing  blackened  fatty  particles,  and  moreover 
the  cleft-like  central  lacteal  in  the  middle  of  each  villus 
will  be  found  to  contain  similar  globules.  Hence  we  infer 
that  the  tatty  matters  are  first  taken  up  from  the  cavity 
of  the  intestine  by  the  columnar  epithelium  cells ;  that 
they  are  transmitted  in  some  way  from  these  to  the 
amoeboid  lymph  cells,  and  that  these  again  convey  them 
to  and  discharge  them  into  the  central  lacteal. 

Preparation  3.  Vessels  of  the  small  intes- 
tine.— The  bloodvessels  of  the  small  intestine  are  to 
be  studied  by  aid  of  vertical  sections  of  the  injected 
gut.  The  sections  may  be  lightly  stained  with 
logwood. 

The  lymphatics  (lacteals)  may  perhaps  be  seen  in  thin 
sections  of  the  uninjected  preparations  as  cleft-like  spaces 
in  the  villi  and  in  the  substance  of  the  mucous  membrane, 
and  surrounding  the  bases  of  the  lymphoid  nodules  which 
make  up  the  Peyerian  patches.  It  is  not  an  easy  matter 
to  inject  those  of  the  mucous  membrane,  although  the 
larger  plexuses  of  the  submucous  and  muscular  coat  can 
be  more  easily  demonstrated. 

Preparation  4.  Nerves  of  the  intestinal 
wall. — The  nerves  of  the  intestinal  canal  form  a 
very  interesting  subject  of  study,  comprising  two  of 
the  closest  and  most  richly  gangliated  plexuses  of 
pale  fibres  which  are  met  with  in  the  animal  body. 
They  may,  moreover,  by  following  the  method  here 
to  be  described,  be  shown  without  any  great  diffi- 


220  PRACTICAL    HISTOLOGY. 

cultj  in  all  parts  of  either  the  small  or  the  large 
intestine.  It  is  preferable  to  choose  an  animal  (e.g. 
rabbit  or  guinea-pig)  in  which  the  intestinal  coats 
are  not  very  thick.  The  following  is  the  mode  of 
procedure:  A  piece  of  glass  tubing  about  a  quarter 
of  an  inch  in  diameter  and  five  or  six  inches  long 
is  taken,  and  one  end  is  drawn  out  into  a  canula, 
whilst  to  the  other  a  small  piece  of  India-rubber 
tube,  furnished  with  a  spring  clip,  is  attached. 
Chloride  of  gold  solution  (J  per  cent.)  is  drawn  up 
into  the  tube  so  as  almost  to  fill  it,  and  the  clip 
is  then  closed,  to  prevent  the  escape  of  the  fluid. 
Care  should  be  taken  not  to  suck  any  of  the 
gold  solution  into  the  mouth.  A  piece  of  intestine 
about  three  inches  long  is  removed  from  the  dead 
animal,  and  if  not  already  empty  its  contents  are 
washed  out  by  a  stream  of  salt  solution.  The  intes- 
tine thus  emptied  and  cleaned  is  ligatured  firmly  at 
one  end,  whilst  into  the  other  is  tied  the  canulated 
end  of  the  glass  tube  containing  the  gold  solution. 
When  thus  secured  the  clip  is  opened  and  the  fluid 
is  allowed  to  flow  into  and  distend  with  moderate 
force  the  piece  of  gut,  the  action  of  gravity  being 
assisted  by  gently  blowing  through  the  India-rubber 
tube.  As  soon  as  the  intestine  is  filled  with  the 
gold  solution  the  clip  is  again  allowed  to  close,  and 
then,  while  an  assistant  holds  the  glass  tube  in  a 
vertical  position,  the  operator  ligatures  the  gut  just 
beyond  the  end  of  the  canula,  which  may  not  be  cut 
away.  The  piece  of  intestine,  thus  filled  with  the 
gold  solution,  is  immersed  for  an  hour  in  more  of 
the  same  liquid.  It  is  then  placed  in  a  dish  of 
water  and  cut  open  longitudinally  with  scissors,  so 
as  to  allow  the  contained  fluid  to  escape,  after  which 
the  puckered,  ligatured  ends  may  also  be  removed. 
The  tissue  being  hardened  by  the  gold  solution,  the 
piece  of  gut  which  remains  retains  its  cylindrical 
shape.  It  is  well  to  halve  it  by  another  longitudinal 
cut,  so  that  both  inner  and  outer  surfaces  may  be 
freely  exposed  to  the  light.  The  pieces  are  now 
placed,  with  their  outer  surfaces  uppermost,  in  a 


NERVOUS  PLEXUSES  OF  INTESTINE.   221 

glass  vessel  of  water  containing  just  enough  acetic 
acid  to  be  sour  to  the  taste,  and  the  vessel  is  covered 
and  allowed  to  stand  in  a  warm  place  freely  exposed 
to  the  sunlight  (see  p.  96).  After  two  days  its  color 
will  be  found  to  have  changed  to  a  dark  violet.  A 
few  drops  of  methylated  spirit  may  then  be  added 
to  the  fluid ;  this  serves  to  aid  the  reduction  of  the 
gold  and  to  prevent  the  growth  of  fungi.  In  another 
day  or  two  the  tissue  will  be  so  dark  as  to  appeal- 
almost  black.  A  portion  is  then  removed  to  a  glass 
dish  of  water  and  prepared  in  the  following  way : 
In  the  first  place,  the  glandular  mucous  membrane 
is  separated  from  the  rest  of  the  intestinal  wall 
either  by  tearing  it  off  with  forceps  or  by  scraping 
it  away  with  the  end  of  a  blunt  scalpel.  There  now 
remain  the  serous  and  two  muscular  layers,  together 
with  the  submucosa.  To  the  inner  surface  of  the 
latter  the  muscularis  mucosae  may  be  still  adherent. 
The  separated  fragments  of  the  mucous  membrane 
are  got  rid  of  by  pouring  away  the  water  first  used 
and  substituting  fresh,  and  then  an  attempt  must 
be  made  by  aid  of  two  pairs  of  forceps,  to  peel  the 
submucosa  off  from  the  inner  surface  of  the  muscular 
coat.  Of  course  if  the  muscularis  mucos?e  has  been 
left,  tluit  will  form  a  part  of  the  layer  which  is  thus 
removed.  The  separation  must  be  done  slowly  and 
carefully,  so  as  to  get  as  large  a  piece  as  possible 
intact.  When  this  is  accomplished  satisfactorily  a 
slide  is  immersed  in  the  wrater,  and  the  portion  of 
submucosa  so  detached  is  floated  on  to  it,  and 
re'moved  from  the  water.  Its  further  preparation 
consists  in  allowing  the  excess  of  water  to  run  off, 
applying  a  cover-glass,  making  sure  first  of  all  that 
the  layer  is  free  from  folds,  and  then  allowing  gly- 
cerine to  pass  under  the  cover-glass  and  replace  the 
water  as  this  evaporates. 

Returning  to  the  remainder  of  the  piece  of  intes- 
tine, the  next  process  consists  in  picking  away  bit 
by  bit  with  forceps  the  comparatively  thick  layer  of 
circular  muscular  fibres.  This  is  not  a  difficult  ro- 


222  PRACTICAL    HISTOLOGY. 

ceeding,  and  when  it  is  finished  all  that  remains  is 
the  thin  serous  coat  and  the  longitudinal  muscular 
layer,  to  the  inner  side  of  which  the  nervous  plexus 
of  Auerbach,  the  intermuscular  plexus,  is  adherent. 
~No  further  separation  is  required,  all  that  is  neces- 
sary being  to  float  the  piece  of  tissue  on  to  a  slide 
with  the  (concave)  inner  surface  uppermost.  But 
before  applying  the  cover-glass  the  preparation  is  to 
be  examined  with  a  low  power,  to  see  that  the  sur- 
face of  the  serous  membrane  is  free  from  a  finely 
granular  precipitate  which  is  apt  to  be  deposited  in 
the  acidulated  water.  If  this  is  present,  the  piece 
must  be  replaced  in  the  water  and  the  precipitate 
gently  brushed  oft'  with  a  soft  camel-hair  pencil. 
The  preparation  is  completed  in  the  same  way  as 
that  of  the  submucosa.  The  latter  shows  Meissner's 
plexus,  the  cords  of  which  are  much  finer  than  those 
of  Auerbach's.  In  both  plexuses  the  nervous  cords 
are  stained  of  a  violet  color  by  the  reduction  of  the 
gold  ;  at  the  points  of  junction  of  the  nervous  cords 
are  groups  of  small  ganglion  cells,  the  nuclei  of 
which  are  hardly  stained  at  all,  and  consequently 
look  clear  in  the  midst  of  the  darkly-stained  cell- 
bodies.  The  distinction  between  the  individual 
cells  is  difficult  to  make  out.  Branches  may  perhaps 
he  traced  passing  from  the  plexus  of  Auerbach 
amongst  the  muscular  fibre-cells :  from  that  of 
Meissner  to  the  muscularis  mucosoe,  if  this  is  present, 
and  perhaps  also  to  the  small  bloodvessels,  which  are 
particularly  well  seen  in  the  preparation  of  the  sub- 
mucous  coat. 

Preparations  5-7.  Large  intestine. — For 
hardening  the  tissue  and  preparing  sections  of  the 
large  intestine  the  same  methods  are  employed  as  for 
the  small  intestine,  so  that  it  is  unnecessary  to  re- 
capitulate them. 

The  injected  large  intestine  is  prepared,  like  the 
stomach,  by  means  of  vertical  and  horizontal  sec- 
tions. 

The  lymphatics  are  not  easy  to  inject,  but  present 
' 'Acuity  than  those  of  the  stomach. 


THE    LIVER.  223 


CHAPTER    XVI. 

THE  LIVER. 

Preparation  1.  ITninjected  liver. — To  prepare 
sections  of  the  liver  small  pieces  are  placed  in  2  per 
cent,  bichromate  of  potash  solution  for  ten  days, 
transferred  from  this  to  weak  spirit,  and  in  twenty- 
four  hours  are  placed  in  strong  spirit,  to  complete 
the  process  of  hardening.  The  tissue  will  be  hard 
enough  to  cut  thin  sections  from  in  another  day  or 
two.  The  sections  are  stained  with  logwood,  and 
mounted  in  dammar  varnish.  They  should  be  made 
in  two  directions,  viz.  (1)  in  a  plane  near  arid  parallel 
to  one  of  the  surfaces  of  the  liver,  and  (2)  vertical  to 
the  surface.  Those  made  in  the  direction  first  named 
will  for  the  most  part  cut  the  central  or  intralobular 
veins  across,  those  in  the  second  direction  may  take 
them  along  their  length ;  the  apparent  arrangement 
of  the  blood  capillaries  and  liver  cells  in  the  indi- 
vidual lobules  will  differ,  both,  in  accordance  with 
this  difference  of  direction  and  also  according  as  the 
lobule  is  cut  exactly  through  its  centre  or  at  some 
part  more  or  less  removed  from  this.  Between  the 
lobules  are  seen  the  branches  of  the  portal  vein, 
always  accompanied  by  a  branch  of  the  bile  duct, 
the  columnar  epithelium  of  which  is  very  well  seen 
in  these  preparations,  and  by  a  branch  of  the  hepatic 
artery.  All  three  are  included  in  a  mass  of  connec- 
tive tissue,  a  prolongation  of  Glisson's  capsule,  en- 
closing them  in  a  so-called  portal  canal.  In  this 
connective  tissue  cleft-like  spaces  may  generally  be 
seen — two  or  three  in  the  section  of  a  portal  canal — 
not  merely  breaks  in  the  connective  tissue,  but  with 
quite  a  definite  wall.  These  are  the  accompanying 


224  PRACTICAL    HISTOLOGY. 

lymphatics.  Other  lymphatics  accompany  the 
branches  of  the  hepatic  veins,  but  are  not  so  easily 
seen  in  the  sections,  although  they  can  be  injected. 
The  branches  of  the  hepatic  veins  are  readily  dis- 
tinguished from  those  of  the  portal  vein,  by  the  fact 
that  they  run  unaccompanied  by  branches  of  the  bile 
duct  and  hepatic  artery.  The  blood  capillaries  of 
the  lobules  look  like  spaces  (tilled  with  round  clear 
bodies,  the  altered  blood  corpuscles)  between  the 
rows  of  cells  (in  the  sections  these  appear  arranged 
simply  in  rows) ;  their  walls  are  very  thin,  and  the 
hepatic  cells  appear  for  the  most  part  to  come  in 
contact  with  the  wall.  But  in  reality  there  is  a 
second  delicate  membrane  around  many  of  the  capil- 
laries, and  between  it  and  the  epithelioid  wall  of  the 
vessel  is  a  space  for  the  passage  of  lymph  (perivascu- 
lar  lymphatic) ;  it  is  difficult  to  make  this  out,  how- 
ever, in  preparations  in  which  the  lymphatics  are 
not  injected.  The  round  nuclei  of  the  liver  cells  are 
deeply  stained  by  the  logwood,  and  the  cells  them- 
selves slightly.  In  the  thinnest  parts  of  the  sections 
the  lines  of  junction  between  neighboring  cells  can 
be  well  made  out,  and  not  unfrequently  the  small 
capillary  passage  for  the  bile  which  intervenes  be- 
tween the  adjacent  sides  of  the  cells  can,  according 
to  the  direction  in  which  it  runs,  be  recognized  with 
a  very  high  power  either  as  a  horizontal  line  or  as  a 
minute  aperture.  To  obtain  the  best  results  the 
pieces  of  liver,  which  are  not  to  be  more  than  an 
inch  or  so  square  and  a  quarter  of  an  inch  thick, 
should  be  placed  in  the  bichromate  solution  quite 
fresh,  from  an  animal  killed  only  a  short  time  pre- 
viously. 

Preparation  2.  Injected  liver. — The  vessels 
of  the  liver  seldom  get  tilled  when  the  rest  of  the 
body  is  injected  from  the  aorta.  It  is  generally 
necessary  to  make  a  special  injection  of  this  organ 
from  the  portal  vein.  For  this  purpose  the  usual 
red  or  blue  gelatine  injection  is  used,  the  apparatus 
being  arranged  as  described  at  p.  14t>.  The  operation 


INJECTION    OF    LIVEK.  225 

is  conducted  as  follows :  The  animal  (rabbit)  having 
been  killed  by  bleeding,1  the  thorax  is  opened  ;  and 
the  pericardium  being  torn  away,  the  heart  is  raised 
and  two  thread  ligatures  are  passed  round  the  inferior 
vena  cava.  One  of  these  is  tightened  as  near  the 
heart  as  possible,  and  then  a  snip  is  made  in  the  vein, 
so  as  to  allow  the  blood  to  escape  freely.  Next,  the 
abdomen  is  opened,  and  the  intestines 'and  stomach 
being  gently  drawn  to  the  left  side,  the  peritoneum 
at  the  back  of  the  abdomen  is  torn  through,  and  a 
ligature  placed  around  the  vena  cava  above  the  ac- 
cession of  the  renal  veins.  The  portal  vein  is  then 
found  in  the  fold  of  peritoneum  which  connects  the 
under  surface  of  the  liver  with  the  stomach,  and  a 
ligature,  in  the  noose  of  which  the  hepatic  artery 
may  be  included,  having  been  passed  round  it  near 
the  liver,  a  snip  is  made  in  the  vessel,  and  the  in- 
jecting canula  is  tied  in.  This  canula  is  now  filled 
by  means  of  a  pipette  with  warm  salt  solution,  and 
the  supply  tube  (from  the  injecting  bottle),  having 
been  completely  filled  by  the  injecting  fluid  to  the 
exclusion  of  air  in  the  same  way  as  in  the  first  in- 
jection (p.  167),  is  slipped  over  the  open  end,  and 
the  injection  at  once  allowed  to  flow.  As  it  passes 
by  the  portal  system  of  veins  through  the  lobules  of 
the  liver  into  the  hepatic  system,  it  forces  whatever 
blood  is  still  contained  in  the  bloodvessels  of  the 
organ  out  into  the  vena  cava,  whence  it  can  freely 
escape  into  the  thorax  through  the  snip  which  was 
there  made  in  the  vein.  As  soon  as  all  the  blood  is 

1  In  injecting  the  whole  body  it  was  recommended  to  kill  the 
animal  by  chloroform.  This  was  for  the  purpose  of  having  the 
bloodvessels  as  much  dilated  as  possible.  When  an  animal  is 
killed  by  bleeding,  the  arteries  contract  very  considerably,  and, 
remaining  contracted  some  little  time  after  death,  offer  a  con- 
siderable resistance  at  first  to  the  passage  of  the  injection,  and 
this  may  tend  to  spoil  the  result  altogether.  In  the  liver,  how- 
ever, the  case  is  different,  since  it  is  not  injected  through  arteries, 
but  through  veins,  which  possess  little  contractility.  Any  blood 
which  remains  in  the  vessels  does  not,  so  long  as  it  remains  fluid, 
impede  the  passage  of  the  injection,  but  it  is  driven  before  it. 


226  PRACTICAL    HISTOLOGY. 

thus  driven  out,  and  only  pure  injecting  fluid  begins 
to  pass,  this  vein  is  occluded  near  the  diaphragm  by 
the  second  thread.  The  pressure  in  the  injecting 
bottle  is  then  slowly  raised,  but  should  not  even  at 
the  utmost  exceed  three  inches  of  mercury,  for  this 
amount  of  pressure  will  cause  all  the  bloodvessels  to 
be  quite  fully  distended,  and  will  effect  a  very  con- 
siderable consequent  enlargement  of  the  organ  ;  more 
might  cause  rupture  and  extravasation.  After  the 
lapse  of  a  few  minutes,  to  allow  of  the  complete  fill- 
ing of  all  the  bloodvessels,  a  second  ligature  is  tied 
round  the  portal  vein  close  to  the  liver  to  prevent 
the  return  of  the  still  fluid  injection,  and  the  canula 
is  cut  out  from  the  portal  vein  (the  pressure  in  the 
apparatus  having  first  been  removed),  and  the  body 
put  into  a  cold  place  so  as  to  permit  the  gelatine  to 
solidify.  It  is  well  to  hasten  the  process  by  pouring 
cold  water — iced  if  possible — over  the  liver.  When 
the  injecting  material  is  entirely  set,  the  organ  is 
removed  and  cut  into  pieces,  which  are  placed  in 
weak  spirit  (half  water)  for  twenty-four  hours.  They 
are  then  put  into  stronger  spirit,  and  in  forty-eight 
hours  more  in  the  strongest.  In  three  or  four  days 
sections  may  be  made  (in  two  directions  as  with  the 
uninjected  organ),  and  mounted,  after  passing 
through  oil  of  cloves,  in  dammar.  It  is  better  not 
to  stain  them. 

During  the  whole  process,  the  greatest  care  must 
be  taken  not  to  handle  the  liver  more  than  can 
possibly  be  helped,  for  it  is  very  readily  scratched 
or  ruptured,  and  any  such  accident  would  tend  to 
permit  the  escape  of  the  fluid  injection.  This  warn- 
ing applies  with  equal,  if  not  greater,  force  to  the 
operation  next  to  be  described ;  that  namely,  of 
filling  the  bile-ducts. 

Preparation  3. — The  bile-ducts  are  injected  with 
Berlin  blue  solution,  2  per  cent.,  the  mercury  appa- 
ratus (Fig.  30)  being  used.  The  solution,  although 
fluid  in  the  cold,  should  nevertheless  be  employed 
warm,  as  it  is  then  less  likely  to  excite  contraction 


LYMPHATICS    OF    THE    LIVER.  227 

of  the  biliary  ducts.  A  rabbit  is  killed  by  bleeding, 
the  abdomen  opened,  and  the  common  bile-duct 
sought  for  close  to  the  portal  vein ;  a  ligature  is 
passed  round  it  and  a  small  piece  of  card  being 
placed  under  as  a  support,  and  to  separate  it  from 
the  accompanying  vessels,  a  snip  is  made  into  it, 
and  a  glass  canula  is  inserted,  and  having  been 
passed  along  the  duct  as  near  to  the  liver  as  possible 
is  tied  in.  The  cystic  duct  is  ligatured  to  prevent 
the  injection  from  passing  into  the  gall-bladder.  In 
the  next  place  the  canula  is  filled  with  warm  Berlin 
blue  solution  by  means  of  a  fine  pipette ;  the  (pre- 
viously filled)  supply  tube  is  attached,  the  clip  on 
this  opened,  and  the  pressure  gradually  raised  to 
about  two  inches  of  mercury.  The  blue  fluid,  driving 
whatever  bile  there  happens  to  be  left  in  the  ducts 
before  it  into  the  lobules,  penetrates  first  into  the 
interlobular  bile-ducts,  and  from  these  into  the  outer 
parts  of  the  lobules,  forcing  the  bile  more  and  more 
towards  the  centre ;  here  of  course  there  is  no  escape 
for  it,  except  that  a  little  may  pass  into  the  lym- 
phatics and  bloodvessels  through  their  walls.  Hence 
it  will  be  understood  that  the  injection  can  only  be 
made  to  fill  the  intercellular  biliary  passages  in  the 
outer  part  of  each  lobule.  The  injection  should  be ' 
persevered  with  for  about  half  an  hour ;  the  bile- 
duct  may  then  be  tied  and  the  injecting  apparatus 
removed ;  after  which  the  liver  is  cut  out  entire, 
without  injuring  it  in  any  way,  and  placed  in  strong 
spirit.  In  twenty-four  hours  it  is  cut  in  pieces,  and 
the  spirit  changed,  and  in  less  than  a  week  the 
pieces  will  be  hard  enough  to  cut.  The  sections 
may  be  stained  slightly  with  logwood. 

Preparation   4.     Lymphatics  of  the   liver. — 

The  lymphatics  of  the  liver  are  injected  through  a  fine 
canula  stuck  obliquety  into  the  superficial  part  of  the 
organ  immediately  beneath  the  capsule.  Either  solution 
of  Berlin  blue  or  alkanet  may  be  used.  The  part  should 
be  quite  fresh.  If  the  injection  be  persisted  in  for  a  long 
while,  the  fluid  may  flow  out  both  by  the  lymphatics 


228  PRACTICAL    HISTOLOGY. 

accompanying  the  portal  vein  and  those  accompanying 
the  hepatic  veins  (Ludwig  and  Fleischl).  Very  fre- 
quently, however,  the  injecting  fluid  finds  its  way  into 
the  blood-system  instead  of  the  lymphatics.  The  injec- 
tion of  the  lymphatics  may  be  accomplished  in  another 
manner,  viz.,  by  seeking  those  lymphatics  which  accom- 
pany the  hepatic  veins  at  the  back  of  the  liver,  and  tying 
a  canula  into  them.  After  a  time  the  fluid  will  be  found 
to  pass  out  by  the  vessels  which  accompany  the  portal 
vein. 

The  investigation  of  the  lymphatics  of  the  liver  is, 
however,  very  difficult,  and  our  knowledge  of  their  course 
and  arrangement  is  still  by  no  means  satisfactory. 

Preparation  5.  Hepatic  cells.— In  addition 
to  what  may  be  learnt  from  sections  of  the  organ, 
teased-out  preparations  afford  much  useful  infor- 
mation, both  of  the  characters  of  the  liver  cells  and 
of  the  connective  tissue  of  the  lobules.  For  this 
purpose  small  portions  of  the  perfectly  fresh  and 
warm  liver  are  broken  up  in  serum  or  salt  solution, 
and  other  portions  are  macerated  for  a  day  or  two 
in  weak  bichromate  of  potash,  and  subsequently 
teased  out  in  water. 


The  pancreas  is  prepared  in  the  same  manner  as 
tbe  salivary  glands,  to  the  description  of  which  the 
student  is  referred. 


THE    SPLEEN.  229 


CHAPTER    XVII. 

THE  SPLEEN  AND  URINARY  ORGANS. 
THE  SPLEEN. 

Preparation  1.    The  uninjected  spleen. — The 

spleen  is  hardened  in  the  same  manner  as  the  liver, 
by  placing  small  pieces  of  it  in  2  per  cent,  solution 
of  bichromate  of  potash,  and  in  about  a  week  or  ten 
days  transferring  them  first  to  weak  and  then  to 
strong  spirit.  The  sections,  which  cannot  be  too 
thin,  are  to  be  stained  deeply  with  logwood,  and 
mounted  by  the  ordinary  mode  of  precedure  in 
dammar  varnish. 

In  these  preparations  the  Malpighian  corpuscles' 
(or  nodules  of  lymphoid  tissue)  are  very  strongly 
colored,  as  are  also  the  trabeculse  which  traverse  the 
pulp,  especially  in  those  animals  in  which  they  are 
largely  composed  of  plain  muscular  tissue ;  the  sub- 
stance of  the  pulp  is  but  slightly  colored  by  the  log- 
wood, only  the  cell-nuclei,  and,  to  a  much  less  extent, 
the  branching  cells  of  the  retiform  tissue  being 
stained.  The  prevailing  color  of  the  pulp  is  yellow- 
ish, owing  to  the  blood  (altered  in  color  by  the  action 
of  the  reagent)  which  at  the  time  of  death  remained 
in  the  interstices  of  the  tissue.  Moreover,  here 
and  there  a  speck  of  coarsely-granular  reddish-yellow 
pigment  may  be  detected,  lodged  in  one  of  the  cor- 
puscles of  the  spleen  pulp.  But  this  will  be  better 
made  out  in  the  teased-out  preparations  subsequently 
to  be  described. 

Preparation  2.     Irrigated   spleen. — By  another 
mode  of  preparing  the  spleen  all  the  blood  is  first  washed 
out  by  a  stream  of  salt  solution,  injected  through  the 
20 


230  PRACTICAL    HISTOLOGY. 

splenic  artery,  and  the  organ  is  then  hardened  in  the 
same  way  as  before.  It  will  much  facilitate  the  process 
of  hardening  if  the  salt  solution  is  followed  by  a  stream 
of  the  bichromate.  This  may  even  be  made  to  distend 
the  organ  somewhat,  the  distension  being  maintained  by 
ligaturing  the  vessels  near  the  hilus.  In  such  a  case  the 
organ  is  to  be  placed  entire  in  2  per  cent,  bichromate,  and 
only  cut  into  pieces  after  forty -eight  hours.  For  a  spleen 
which  has  been  thus  prepared,  the  cacao-butter  method 
of  embedding  may  be  employed,  after  a  thin  piece  has 
been  stained  by  alcoholic  logwood.  By  thus  removing 
the  blood  corpuscles  the  retiform  tissue  of  the  pulp  is 
better  seen.  Klein  (Quarterly  Journal  of  Microsco- 
pical Science,  October,  1875  ,  relying  on  appearances 
presented  by  preparations  made  in  this  way,  describes 
the  retiform  tissue  of  the  spleen  as  entirely  made  up  of 
flattened  cells  forming  by  their  junction  a  "honeycomb 
of  membranes."  That  such  a  description  is  far  too  ex- 
clusive, the  study  of  teased  preparations  amply  demon- 
strates, for  the  network  of  branched  cells,  described  by 
almost  all  previous  observers,  is  readily  seen  in  them 
(see  below,  Prep.  4). 

Preparation  3.  Injected  spleen.— The  spleen 
may  have  been  injected  in  the  animal  which  was 
injected  entire  ;  if  this  is  not  the  case,  a  special  in- 
jection is  to  be  made  from  the  splenic  artery.  When 
successfully  accomplished,  the  vessels  are  as  usual 
ligatured  to  prevent  the  escape  of  the  injection,  and 
the  organ  is  immersed  entire  in  spirit,  at  first  weak 
but  with  the  strength  afterwards  gradually  increased, 
as  in  the  case  of  the  liver.  The  sections  will  show 
what  at  first  sight  look  like  accidental  extrava- 
sations, large  patches  namely  of  injection  distributed 
all  over  the  organ,  with  the  exception  of  large  round 
white  patches  here  and  there,  pervaded  by  a  few 
capillaries.  The  white  patches  are  sections  of  the 
Malpighian  corpuscles,  and  the  part  permeated  by 
the  injection  is  of  course  tbe  pulp,  into  which  the 
arterial  capillaries  freely  open. 

Preparation  4.  Splenic  cells.  —  To  obtain 
specimens  of  the  spleen  substance,  which  will  show 


THE    KIDNEYS.  231 

in  a  separated  condition  the  cellular  elements  which 
it  contains,  and  of  which  it  is  composed,  a  small 
portion  of  the  fresh  organ  may  be  teased  out  with 
needles  in  a  little  salt'solution  or  serum.  But  it 
will  be  found  that  so  much  blood  is  incorporated 
with  the  spleen  substance  (it  forms,  in  fact,  most  of 
the  soft  matter  which  can  be  expressed  from  the 
fresh  section)  that  the  view  of  the  other  parts  is 
obscured  by  innumerable  red  blood  corpuscles. 
Hence  before  teasing  a  piece  it  should  be  placed  for 
forty-eight  hours  in  J  per  cent,  bichromate  of  potash 
solution.  This  destroys  the  red  corpuscles  whilst 
preserving  the  character  of,  and  at  the  same  time 
macerating  somewhat  the  proper  substance  of  the 
spleen,  so  "that  the  cells  are  now  readily  separated 
and  seen.  By  far  the  greater  number  are  lymph 
corpuscles  from  the  lymphoid  tissue  of  the  Malpig- 
hian  nodules  and  of  the  arterial  adventitia.  But, 
besides  these,  other  cells  are  met  with ;  larger,  often 
flattened,  and  many  of  them  with  fine  branchings. 
These  are  cells  of  the  retiform  tissue  ;  some  of  them 
contain  pigment  granules,  as  already  intimated. 
They  may  be  found^either  entirely  isolated,  or  form- 
ing a  fine  network  by  the  intercommunication  of 
their  branches.  Their  nuclei,  as  well  as  those  of  the 
lymphoid  cells,  are  well  brought  out  if  a  little  log- 
wood solution  is  permitted  to  pass  under  the  cover- 
glass.  In  the  fresh  preparations,  not  treated  by  bi- 
chromate of  potash  or  any  other  reagent,  but  made 
in  serum,  some  of  the  cells  may  perhaps  be  found 
containing  red  blood  corpuscles  in  their  interior,  and 
transitions  from  these  to  those  containing  pigment 
are  met  with. 

THE  KIDNEY. 

Preparation  5.    The  uninjected  kidney. — The 

kidney  is  hardened  in  the  same  way  as  the  liver  and 
spleen,  viz.,  by  a  strong  solution  of  bichromate  of 
potash  (two  per  cent.).  The  piece  or  pieces  that  are 


232  PRACTICAL    HISTOLOGY. 

employed  should  include  both  cortical  and  medul- 
lary parts;  but  at  the  same  time  should  not  be 
thicker  than  from  a  quarter  of  an  inch  to  half  an 
inch,  otherwise  the  preservative  fluid  will  not  pene- 
trate rapidly  enough  to  the  deeper  parts.  They  are 
to  remain  in  the  bichromate  solution  (a  relatively 
large  quantity)  for  three  weeks;  are  then  placed  in 
weak  spirit,  and  in  twenty -four  hours  transferred  to 
strong  spirit.  In  three  or  four  days  more  the  pieces 
are  firm  enough  to  cut.  They  will  perhaps  be  large 
enough  to  hold  in  the  hand,  and  thus  the  necessity 
of  imbedding  will  be  avoided;  sections  as  thin  as 
possible  are  to  be  made  in  a  plane  vertical  to  the 
surface  of  the  organ,  and  large  enough  to  include 
both  cortical  substance  and  Malpighian  pyramid. 
One  such  section  is  first  -transferred  from  the  spirit 
to  water,  and  is  then  simply  mounted  in  glycerine; 
another  is  stained  with  logwood,  and  after  treatment 
by  the  customary  process  is  mounted  in  dammar 
varnish.  These  sections  will  show:  in  the  cortical 
substance  the  Malpighian  corpuscles,  the  convoluted 
tubules  variously  cut,  and  the  prolongations  of  the 
straight  tubules  of  the  medullary  substance  and  of 
the  tubules  of  Henle  ;  in  the  pyramidal  part  the  two 
last-named  tubules,  and  the  collecting  and  excretory 
tubules,  seen  longitudinally,  with  a  large  number  of 
bloodvessels  running  parallel  to  and  between  them. 

Preparation  6. — A  transverse  section  also  should 
be  obtained  of  the  medullary  substance.  With  this 
object  the  end  of  a  pyramid  is  cut  off  with  a  razor, 
and,  as  it  is  too  small  to  hold  in  the  hand,  it  is  im- 
bedded in  wax-mass  in  such  a  position  that  the 
tubules  will  be  cut  exactly  across.  Sections  from 
this  are  mounted  like  the  others  in  glycerine  and  in 
dammar  varnish. 

The  kidney  of  the  dog  or  of  almost  any  other  ani- 
mal may  be  used  for  the  above  preparations,  and  has 
the  advantage  over  the  human  organ,  that  it  is  ob- 
tainable in  a  fresher  condition.  But  since  the  epi- 
thelium of  the  tubules,  at  all  events  of  the  convoluted 


THE    KIDNEYS.  233 

tubules,  differs  somewhat  in  appearance  in  the  human 
kidney  as  compared  with  that  of  the  lower  animals, 
a  portion  of  a  perfectly  healthy  organ  should  be  pro- 
cured from  the  post-mortem  room  as  fresh  as  possible, 
and  prepared  for  observation  in  the  same  manner. 

Preparation  7.  The  injected  kidney.— The 
bloodvessels  of  the  kidney  will  very  probably  be 
filled  in  injecting  an  animal  entire ;  but,  if  this 
should  not  have  been  the  case,  it  is  not  difficult  to 
make  a  special  injection  of  the  separated  organ  from 
the  renal  artery.  The  red  gelatine  injection  may  be 
used,  and  the  kidney  is  kept -warm,  and  the  injection 
maintained  for  a  considerable  time,  in  order  that  the 
vessels  of  the  glomeruli  and  the  network  of  capilla- 
ries in  the  cortical  substance  supplied  by  their  effer- 
ent vessels  may  be  completely  tilled.  The  organ  is 
then  set  aside  in  a  cool  place  (surrounded  by  ice,  if 
possible),  and,  when  the  gelatine  is  completely  set, 
is  cut  into  three  or  four  pieces  and  hardened  gradu- 
ally, as  usual,  with  alcohol.  The  sections,  which 
need  not  be  very  thin,  but  should  be  quite  even,  and 
comprise  the  whole  thickness  of  the  organ,  are  to  be 
mounted,  unstained,  in  dammar  varnish. 

Preparation  8.  Uriniferous  tubules  — The  uri- 
mferous  tubules  may  be  injected  from  the  ureters  for  a 
considerable  part  of  their  length  simultaneously,  if  it  be 
desired,  with  the  injection  of  the  bloodvessels  by  a  solu- 
tion of  Berlin  blue.  But  even  if  well  filled  they  are  too 
densely  arranged  to  render  it  possible  to  trace  individual 
tubules  along  their  whole  extent.  This  may  be  better 
accomplished  by  making  teased  preparations  of  the  kid- 
neys of  small  animals,  which  have  undergone  some  pro- 
cess of  preparation,  having  Cor  its  object  the  solution  or 
softening  of  the  intertubular  substance.  Several  such 
processes  have  been  proposed,  but  none  yield  entirely 
satisfactory  results.  The  best,  perhaps,  that  has  yet  been 
tried  consists  in  digesting  tolerably  thick  slices  of  a  small 
kidney  in  a  mixture  of  four  parts  of  spirit  and  one  of 
hydrochloric  acid,  kept  boiling  fur  three  or  four  hours. 
The  boiling  is  performed  in  a  flask  fitted  with  a  cork, 
through  which  M  louo-  vertical  tube  passes;  in  this  much 

20* 


234  PRACTICAL    HISTOLOGY. 

of  the  vapor  which  is  driven  off  by  the  boiling  becomes 
condensed,  and  flows  down  again  into  the  flask.  After 
the  time  mentioned  the  slices  are  removed  and  placed  in 
water;  and,  after  lying  in  this  for  a  few  days,  minute 
shreds,  comprising  the  whole  depth  from  external  surface 
to  papillae,  are  split  off'  with  needles,  placed  on  a  slide, 
and  unravelled  as  much  as  possible  by  aid  of  the  dissect- 
ing microscope.  The  preparation  is  covered  with  a  spe- 
cially large  piece  of  covering-glass  (a  hair  being  first 
added  to  avert  the  pressure  of  the  glass  on  the  soft 
tubules),  and  stained  by  drawing  picric  acid  solution 
under  the  cover-glass.  This  soon  colors  the  tissue  in- 
tensely yellow;  glycerine  may  then  be  allowed  to  pass  in 
at  the  border  in  order  to  complete  the  preparation.  Some 
of  the  tubules  will  be  found  isolated  for  a  considerable 
part  of  their  length,  and  the  passage  of  the  convoluted 
tubules  into  the  looped  tubes  of  Henle  may  especially  be 
well  seen.  The  epithelium  of  the  tubules  is  for  the  most 
part  well  preserved,  but  that  of  the  convoluted  ones  has 
become  very  granular,  and  so  swollen  out  as  to  completely 
fill  up  the  tubules. 

Preparation  9.  Examination  of  the  fresh 
kidney. — When  by  these  various  means  sufficient 
acquaintance  ha*s  been  gained  with  the  various 
tubules  and  their  contents  in  situ,  the  examination 
of  the  fresh  tissue  in  serum  may  be  attempted. 
With  this  object  small  snips  are  to  be  made  from 
different  parts  of  a  freshly-cut  surface  with  a  pair  of 
curved  scissors,  and  teased  out  in  a  drop  of  serum, 
with  the  aid  of  the  dissecting  microscope,  so  as  to 
separate  as  many  of  the  tubules  as  possible.  In 
doing  this  much  of  the  epithelium  will  become 
detached,  and  the  characters  of  the  individual  cells 
in  the  fresh  condition  may  be  studied. 

Preparation  10. — The  epitheloid  cells  of  the  capsules 
of  Bowman  and  of  the  basement  membranes  of  the  tubules 
may  be  shown  by  nitrate  of  silver.  For  this  purpose  a 
fresh  kidney  is  sliced  in  half  by  a  single  cut  with  a  sharp 
razor  in  the  direction  of  the  tubules.  One  of  the  halves 
is  thoroughly  washed  with  distilled  water,  and  solution 
of  nitrate  of  silver  (one-half  per  cent.)  is  poured  over  the 


THE    URETERS.  235 

cut  surface.  A  fter  a  minute  and  a  half  the  silver  solution 
is  rinsed  off  with  distilled  water,  and  the  piece  of  kidney 
is  placed  in  a  beaker  of  strong  spirit,  with  the  silvered 
surface  exposed  to  the  sunlight.  When  brown  it  may  be 
removed  from  the  light,  but  is  left  in  the  spirit  for  twenty- 
four  hours;  one  or  two  sections  are  then  made  from  the 
brown  surface,  clarified  in  oil  of  cloves,  and  mounted  in 
dammar. 

THE  SUPRARENAL  CAPSULE. 

Preparation  11. — To  prepare  the  suprarenal  cap- 
sule it  is  separated  from  the  surrounding  fat,  divided 
into  two  or  three  pieces  by  transverse  cuts,  and  placed 
in  two  per  cent,  of  bichromate  of  potash  solution  for 
fourteen  days,  when  the  hardening  may  be  completed 
in  spirit  in  the  usual  manner.  Hardening  the  organ 
in  spirit  alone  also  gives  very  good  results.  The 
mode  of  preparing  the  sections,  which  should  include 
both  cortical  and  medullary  substance,  calls  for  no 
special  description. 

If  the  human  suprarenal  is  not  obtainable  in  a 
fresh  condition  (the  medullary  substance  very  readily 
softens  and  breaks  down  after  death),  that  of  the 
guinea-pig,  which  is  large  comparatively  to  the 
size  of  the  animal,  and  has  the  distinction  between 
cortical  and  medullary  substance  well  marked,  may 
advantageously  be  employed. 

Preparation  12. — In  a  teased-out  preparation  of 
the  fresh  organ  the  cellular  elements  of  the  cortical 
and  medullary  substance  may  respectively  be  studied, 
and  the  effect  of  a  solution  of  yellow  chromate  of 
potash  in  coloring  the  medullary  cells  brown  may 
be  observed. 

THE  URETERS. 

Preparation  13. — The  ureters  are  prepared  in 
the  same  way  as  the  intestine — by  moderately  dis- 
tending an  excised  portion  with  a  mixture  of  equal 
parts  of  spirit  arid  |  per  cent,  chromic  acid  solution, 


236  PRACTICAL    HISTOLOGY. 

and  placing  the  piece  thus  distended  in  a  beaker 
containing  some  of  the  same  mixture.  After  twenty- 
four  hours  the  tube  is  slit  open,  and  transferred  to 
spirit  for  two  or  three  days.  The  sections  are  to  be 
made  across  the  length  of  the  tube,  and  stained  and 
mounted  in  the  ordinary  manner. 

Preparation  14,  Epithelium  of  ureter. — To 
study  the  separated  epithelial  cells  a  piece  as  fresh 
as  possible  is  cut  open,  pinned  out  on  a  cork  with 
the  inner  surface  uppermost,  and  immersed  in  J  per 
cent,  bichromate  of  potash  solution  for  from  twenty- 
four  to  forty-eight  hours.  Some  of  the  epithelium 
is  then  scraped  off  with  a  spear-shaped  needle  or  the 
end  of  a  scalpel,  and  is  broken  up  in  a  drop  of  water. 
After  the  addition  of  a  piece  of  hair  to  the  fluid 
the  cover-glass  may  be  applied,  and  the  preparation 
examined  with  a  high  power.  If  it  prove  successful, 
with  many  of  the  epithelial  cells  fully  separated,  it 
may  be  permanently  preserved.  With  this  object 
the  cells  should  first  be  stained,  by  allowing  weak 
logwood  solution  to  run  under  the  edge' of  the  cover- 
glass.  The  logwood  is  to  be  followed  by  a  drop  of 
glycerine  applied  at  the  same  edge;  and,  when  the 
glycerine  has  become  diffused  underneath,  all  that 
is  necessary  is  to  cement  the  cover-glass. 

THE  BLADDER. 

The  urinary  bladder,  both  for  sections  and  teased- 
out  preparations,  is  prepared  by  exactly  the  same 
methods  as  the  ureters.  To  distend  it  a  glass  canula, 
connected  by  an  India-rubber  tube  with  a  bottle 
containing  the  chromic  fluid,  is  tied  into  the  urethra. 
The  organ  must  not  be  over-distended,  but  only 
moderately  filled.  Any  urine  which  it  may  contain 
should  first  be  allowed  to  run  out  through  the 
canula. 


THE    GENERATIVE    ORGANS.  237 


CHAPTER   XVIII. 

THE  GENERATIVE  ORGANS. 

Preparation  1. — Those  parts  which  contain  erec- 
tile tissue  will  be  best  studied  after  having  been 
injected.  Their  bloodvessels  and  sinuses  may  have 
been  tilled  in  the  animal  which  was  injected  entire 
from  the  root  of  the  aorta;  but,  if  not,  a  special 
injection  from  the  lower  end  of  the  abdominal  aorta 
is  to  be  made,  the  arteries  supplying  the  lower  limbs 
being  first  tied  to  prevent  waste  of  the  injection. 
The  hardening  of  the  parts  in  spirit  must  be  effected 
very  gradually  (the  spirit  being  daily  made  stronger), 
since  otherwise  the  gelatine  shrinks  away  from  the 
walls  of  the  venous  sinuses,  and  the  preparation 
becomes  in  great  measure  spoiled.  The  sections 
which  are  made  should  some  of  them  be  mounted, 
unstained,  in  dammar,  others  after  being  lightly 
stained  with  logwood,  so  that  the  plain  muscular 
and  fibrous  tissue,  and  also,  in  sections  including 
the  urethra,  the  epithelium  of  that  tube  may  be 
exhibited  as  well  as  the  vessels. 

Preparation  2. — Parts  which  have  not  been 
injected  are  hardened  in  2  per  cent,  bichromate  of 
potash  solution  (fourteen  days),  or  £  per  cent,  chromic 
acid  (seven  days),  followed  by  spirit  in  either  case. 

Preparation  3. — The  glandular  organs,  such  as 
the  prostate  and  vesiculse  seminales,  are  prepared 
either  with  the  £  per  cent,  chromic  acid  solution 
followed  by  spirit,  or  with  spirit  alone.  The  subse- 
quent processes  of  staining  and  mounting  are  the 
same  for  all,  except  that  it  will  be  found,  as  a  rule, 
that  those  which  have  been  in  chromic  acid  stain 


238  PRACTICAL    HISTOLOGY. 

less  readily  than  those  which  have  simply  heen 
hardened  in  spirit. 

Preparations  4-6. — The  scrotum,  and  labia,  and 
the  vagina,  are  prepared  in  the  same  way  as  the 
skin  (see  p.  187). 

Preparation  7. — The  human  uterus  is  best 
hardened  in  the  2  per  cent,  bichromate  of  potash  ; 
its  cavity  should  be  freely  laid  open.  In  animals 
(the  rabbit,  for  instance),  where  it  is  more  membra- 
nous, the  uterus  and  the  upper  part  of  the  vagina 
may  be  prepared  together  by  distending  them  with 
the  spirit  and  J  per  cent,  chromic  acid  mixture 
through  a  canula  tied  into  the  lower  part  of  the 
vagina.  The  vagina  is  then  tied,  and  the  organs 
are  cut  out  and  placed  in  a  quantity  of  the  solution  ; 
in  twenty-four  hours  they  are  laid  open  and  the 
fluid  renewed,  and  in  another  day  or  two  are  ready 
to  be  put  into  spirit.  The  sections  are  stained  with 
logwood,  and  mounted  in  dammar. 

Preparation  8.  Section  of  ovary. — The  ova- 
ries are  prepared  by  placing  them — with  as  little 
handling  as  possible,  so  as  to  avoid  rubbing  off  the 
columnar  epithelium  which  covers  the  surface — in 
£  per  cent,  chromic  acid  (whole  if  taken  from  a 
email  animal,  such  as  a  rabbit  or  cat;  cut  into  two 
or  three  pieces  if  from  a  larger  one).  In  most  of 
the  lower  animals  they  must  be  sought  much  higher 
in  the  abdomen  than  in  the  human  female;  in  the 
rabbit  they  occur  as  small  elongated  bodies,  dotted 
all  over  with  little  projections  (the  Graatian  follicles), 
and  situated  just  below  the  kidneys.  They  are 
left  in  the  chromic  solution  for  seven  days,  and  then 
placed  in  spirit,  and  in  two  or  three  days  more  are 
ready  for  cutting.  The  hardening  is  effected  still 
more  readily  by  using  the  chromic  and  spirit  mix- 
ture. The  sections  are  to  be  stained  in  carmine 
solution  ;  for  logwood  sometimes  colors  very  deeply 
the  coagulated  fluid  in  the  Graafian  follicles,  so  that 
the  epithelial  contents  are  obscured  ;  this  coloration 
is  probably  owing  to  the  presence  of  mucus  (or  rather 


THE    OVUM.  239 

mucin)  in  the  secretion  of  the  follicle.  After  having 
been  stained  the  sections  are  passed  through  alcohol 
and  oil  of  cloves,  and  mounted  in  dammar. 

Preparation  9.  The  ovum.— The  ripe  mam- 
malian ovum,  although  it  can  be  seen  within  the 
larger  Graafian  follicles  in  the  sections  of  the 
hardened  organ,  forms  a  much  more  beautiful  ob- 
ject when  obtained  isolated  from  the  ovary  of  a 
recently-killed  animal.  A  full-grown  doe  rabbit, 
not  pregnant,  is  to  be  sacrificed  for  this  purpose. 
One  of  the  ovaries  having  been  removed,  it  is  held 
firmly  between  the  finger  and  thumb  over  a  clean 
glass  slide,  in  such  a  position  that  the  largest  and 
most  prominent  Graafian  follicle  is  almost  in  con- 
tact with  the  middle  of  the  slide.  The  follicle  is 
then  picked  with  a  sharp-pointed  scalpel,  so  as  to 
cause  the  fluid  contents  of  the  follicle  to  spirt  out, 
carrying  with  them  the  ovum,  surrounded  by  a 
number  of  the  epithelium  cells.  The  ovum  is  rather 
too  small  to  be  detected  with  the  naked  eye,  but  its 
presence  may  be  suspected  if,  on  glancing  at  the 
slide  in  such  a  manner  that  the  light  is  reflected 
from  the  surface  of  the  fluid  to  the  eye,  a  slight  pro- 
minence is  observed  on  the  otherwise  flat  surface. 
Its  presence  here  is  confirmed  by  examination  with 
a  low  power,  and  it  may  then  be  carefully  observed 
with  the  ordinary  high  power.  It  is  better,  if  pos- 
sible, not  to  apply  a  cover-glass,  for  the  zona  pellu- 
cida  is  apt  to  become  broken  ;  and,  moreover,  even 
slight  pressure  spoils  in  great  measure  the  natural 
appearance  of  the  object  But  if  the  objective 
becomes  dimmed  by  its  proximity  to 'the  fluid,  or  if 
it  is  desired  to  employ  an  immersion,  then  a  thin 
cover-glass  must  be  used,  and  to  protect  the  ovum 
from  pressure  a  narrow  slip  of  thick  paper  (an  ordi- 
nary hair  is  not  thick  enough)  is  to  be  put  on  either 
side  before  the  cover-glass  is  applied.  If  the  fluid 
which  accompanies  the  ovurn  from  the  Graafian 
follicles  is  not  in  sufficient  quantity,  a  drop  of  aque- 


240  PRACTICAL    HISTOLOGY. 

ous  humor  may  be  added  to  it.     It  is  not  possible  to 
preserve  this  preparation  permanently. 
Preparation  10.    Artificial  impregnation. — 

If  a  preparation  similar  to  that  just  described  is  ex- 
amined on  the  warm  stage  at  the  temperature  of  the 
body,  and  a  little  of  the  seminal  secretion  of  a  male 
rabbit  with  the  spermatozoa  in  full  activity  is  added, 
the  penetration  of  these  into  the  zona  pellucida  ot" 
the  ovum  can  be  observed. 

Preparation  11.  Section  of  uninjected  testis. 
— The  testis  is  one  of  the  most  difficult  organs  to 
prepare,  owing  to  the  looseness  of  its  structure.  It 
is  firmest  in  those  animals  (cat,  pig)  in  which  the 
peculiar,  granular,  polyhedral  cells,  which  accom- 
pany and  surround  the  bloodvessels,  are  most  nume- 
rous. To  harden  it  two  or  three  cuts  are  made 
almost  through  its  whole  thickness,  and  it  is  then 
placed  for  ten  days  in  2  per  cent,  bichromate  of  pot- 
ash solution,  and  afterwards  in  spirit.  Sections  of 
both  the  body  of  the  testis  and  the  epididymis  are 
to  be  made. 

Preparation  12.  Lymphatics  of  testis. — In 
the  sections  made  as  above  there  will  be  observed  in 
the  interstices  between  the  seminiferous  tubules 
large,  cleft-like  spaces,  looking  almost  like  acciden- 
tal clefts  in  the  loose  connective  tissue  uniting  the 
tubules.  They  are  in  reality,  however,  the  lacunar 
commencements  of  the  lymphatics.  To  show  this 
the  following  simple  experiment  may  be  performed : 
In  a  recently-killed  dog  the  fine  canula  of  a  Pravaz 
syringe,  filled  with  Berlin  blue  solution,  is  stuck 
through  the  scrotum  into  the  middle  of  the  sub- 
stance of  one  of  the  testes,  and  the  solution  is  slowly, 
and  without  exerting  any  considerable  pressure,  in- 
jected into  the  organ.  If  the  abdomen  is  opened  the 
blue  fluid  will  soon  be  seen  passing  along  the  lym- 
phatics which  run  in  the  spermatic  cord,  and  from 
these  into  those  of  the  back  of  the  pelvis  and  abdo- 
men, at  length  reaching  the  thoracic  duct.  If  now 
the  testis  is  removed  and  hardened  in  spirit,  and 


TUNICA    VAGINALIS.  241 

sections  are  made  of  the  hardened  organ,  it  will  be 
found  that  the  intertubular  spaces  are  occupied  by 
the  blue  substance,  and,  since  they  are  proved  by 
the  injection  to  be  in  free  communication  with  the 
lymphatics  which  leave  the  organ,  the  spaces  are 
to  be  looked  upon  as  giving  origin  to  the  lymphatics. 

Preparation  13.  Isolation  of  the  seminife- 
rous tubules. — For  obtaining  the  tubules  isolated 
for  a  considerable  length,  pieces  of  the  testis  (pre- 
ferably human)  are  placed  for  a  day  or  two  in 
hydrochloric  acid,  diluted  with  -J  its  volume  of 
water,  and  maintained  at  30°  C.  They  are  then 
allowed  to  lie  in  water  until  the  tubules  can  be 
readily  separated  with  needles  (Mihalkovics,  Lud- 
wig's  "Arbeiten,"  1874).  Teased-out  preparations 
of  the  fresh  testis-substance  are  also  to  be  made  in 
serum,  to  exhibit  the  form,  stages  of  development, 
and  movements  of  the  spermatozoa.  For  the  object 
last  named  the  preparation  should  be  examined  on 
the  warm  stage. 

Preparation  14.  Epithelioid  cells  of  semi- 
niferous tubules. — To  exhibit  the  fact  that  the 
apparently  structureless  basement  membrane  of  the 
seminiferous  tubules  is  in  reality  composed  of  layers 
of  flattened  epithelioid  cells,  a  portion  of  the  testi- 
cular  substance  is  partially  unravelled  in  distilled 
water,  and  some  of  the  tubules  which  are  thus 
isolated  are  dipped  into  nitrate  of  silver  solution 
for  a  minute,  and,  after  being  again  rinsed  in  water, 
are  mounted  in  glycerine  and  exposed  to  the  light ; 
the  lines  of  junction  between  the  flattened  cells  are 
by  this  means  made  evident. 

Preparations  15,  16.  Tunica  vaginalis.— The 
tunica  vaginalis  is  to  be  prepared  in  the  same  way 
as  the  other  serous  membranes  (with  nitrate  of 
silver),  partly  unbrushed  to  show  the  epithelioid 
covering,  and  partly  brushed  tor  the  sake  of  exhibit- 
ing the  parts  beneath.  For  the  preparation  of  the 
visceral  part,  the  process  is  similar  to  that  adopted 
for  the  pericardium  covering  the  surface  of  the  heart 
21 


242  PRACTICAL    HISTOLOGY. 

(p.  193),  and  need  not  here  be  more  particularly  de- 
scribed. 
Preparation  17.     The    mammary  glands,— 

Small  pieces  of  these  organs  are  hardened  by  being 
placed  in  ^  per  cent,  chromic  acid  solution  for  a 
week  or  ten  days,  subsequently  transferring  them 
to  spirit.  The  mixture  of  equal  parts  of  spirit  and 
J  per  cent,  chromic  acid  may  also  be  used  and  pro- 
duces the  desired  result  more  rapidly  (in  two  or 
three  days);  moreover  the  sections  stain  more  readily 
with  logwood.  They  may  be  mounted  either  in 
dammar  or  in  glycerine.  The  appearances  presented 
by  the  cells  of  the  alveoli  vary  considerably  accord- 
ing to  the  state  of  functional  activity  of  the  gland. 
(See  Creighton,  Report  of  the  Medical  Officer  to  the 
Privy  Council,  1875,  and  Journal  of  Anatomy  and 
Physiology,  October,  1876.) 


THE    BRAIN    AND    SPINAL    CORD.  243 


CHAPTER    XIX. 

THE  CENTRAL  NEVOUS  SYSTEM  ;  THE  BRAIN  AND 
SPINAL  CORD. 

SOME  experience  has  already  been  obtained  of  tbe 
methods  which  are  employed  for  studying  the  cellu- 
lar elements  of  the  central  nervous  system  in  an 
isolated  condition  (see  p.  130).  The  spinal  cord  was 
the  part  then  under  investigation,  but  the  nerve- 
cells  found  in  the  gray  matter  of  the  cerebrum  and 
of  the  cerebellum  may  be  observed  in  the  same 
manner  with  equally  satisfactory  results.  Without 
delaying,  then,  to  repeat  the  directions  there  given, 
we  may  pass  on  to  describe  the  best  methods  of  pre- 
paring sections  of  the  parts  in  question. 

Preparation  with  bichromate  of  ammonia. — 
To  harden  any  part  of  the  central  nervous  system 
the  most  generally  useful  reagent  is  the  bichromate 
of  ammonia  (2  or  3  per  cent,  solution).  This  will  of 
itself  render  the  tissue  sufficiently  firm  for  obtain- 
ing thin  sections,  but  it  is  always  best  to  finish  first 
with  weak  and  then  with  strong  spirit,  especially  as 
this  fluid  must  be  used  to  wet  the  razor.  The  pieces 
to  be  hardened  should  not  be  too  large,  or  at  all 
events  not  to  thick,  but  the  solution  has  consider- 
able power  of  penetration  (differing  in  this  respect 
from  chromic  acid),  and  the  whole  length  of  the 
spinal  cord  of  any  of  the  smaller  quadrupeds,  and 
even  that  of  man,  may  be  hardened  in  it  intact  if 
put  in  perfectly  fresh.  It  is  always  better,  however, 
to  cut  it  into  short  lengths.  The  pieces  are  ready 
to  be  transferred  from  the  bichromate  of  ammonia 
solution  to  dilute  spirit  in  three  or  four  days  if 
small ;  in  a  week  if  rather  larger:  after  twenty-four 
hours  they  are  placed  in  strong  spirit. 


244  PRACTICAL    HISTOLOGY. 

In.  this  way  a  piece  of  the  cerebellum  two  or  three 
small  pieces  from  different  parts  of  the  convoluted 
surface  of  the  cerebrum,  the  medulla  oblongata,  and 
pieces  of  the  spinal  cord  from  the  middle  of  the  three 
regions  (cervical,  dorsal,  and  lumbar)  are  to  be  pre- 
pared. They  should  be  taken  from  the  human  sub- 
ject if  possible,  although  it  is  useful  to  study  the 
parts,  especially  the  spinal  cord  and  medulla,  in  the 
lower  animals  as  well.  The  sections,  at  least  those 
of  the  spinal  cord  and  medulla,  may  be  made  with 
the  aid  of  a  microtome  (see  Appendix),  and,  after 
being  stained,  are  treated  in  the  usual  manner  with 
alcohol  and  oil  of  cloves,  and  mounted  in  dammar 
varnish.  Logwood  may  be  used  for  the  purpose  of 
staining  the  sections,  and  also  carmine;  but,  on  the 
whole,  for  sections  of  the  central  nervous  system, 
some  of  the  aniline  dyes  (which  may  for  the  most 
part  be  employed  in  either  alcoholic  or  watery  solu- 
tion) give  better  results.  One  of  the  best  is  that 
known  in  commerce  as  aniline-blue-black,  which 
stains  the  nerve-cells  and  the  axis-cylinders  of  a  dark 
slate-blue  color.  For  the  cerebellum  a  solution  of 
aniline  blue  is  recommended. 

A  double  coloration  by  logwood  and  eosine  (an  aniline 
dye,  possessing  a  red  color)  has  been  recently  advocated. 
The  eosine  is  to  be  used  in  solution  in  alcohol  (see  Appen- 
dix). The  sections  are  first  stained  with  logwood,  and 
then  placed  in  eosine.  When  sufficiently  colored  by  this 
they  are  passed  through  oil  of  cloves  and  mounted  in 
dammar. 

Preparation  by  Sankey's  method, — For  the 

preparation  of  large  sections  of  different  parts  of  the 
brain  (particularly  of  the  cerebral  convolutions  and 
of  the  cerebellum),  destined  more  particularly  to 
show  the  nerve-cells  and  the  course  and  connection 
of  their  offsets,  there  is  no  better  method  than  that 
described  by  Sankey  (''Quarterly  Journal  of  Micro- 
scopical Science,"  April,  1876).  It  is  shortly  as  fol- 
lows: With  a  large  and  long  knife  wetted  with 
spirit,  slices  of  the  fresh  brain  (of  the  cerebellum 


THE    BRAIN    AND    SPINAL    CORD.  245 

across  the  laminae,  for  example)  are  made,  as  thin  as 
possible  under  the  circumstances — one-tenth  of  an 
inch  thick  perhaps — and  are  placed  in  a  strong 
watery  solution  (seven  per  cent.)  of  the  aniline-blue- 
black.  After  three  hours  the  staining  solution  is 
poured  off  and  water  substituted,  and  in  a  few 
minutes  this,  which  will  still  be  intensely  colored, 
is  replaced  by  fresh  water,  and  so  on  until  the  excess 
of  the  staining  fluid  is  washed  away  from  the  slices. 
If  one  is  cut  in  half  it  will  be  seen  that  only  the  sur- 
faces are  stained,  the  aniline  solution  not  having 
penetrated  to  the  interior.  When  the  washing  is 
completed  the  slices  are  transferred  from  the  water 
to  glass  slides.  This  can  be  effected,  without  risk 
of  breaking  the  sections,  by  floating  them  on  to  an 
immersed  slide,  and  raising  both  together  out  of  the 
water.  The  excess  of  water  is  then  allowed  to  drain 
off',  and  the  pieces  are  left  in  a  dry  place  exposed  to 
the  air,  so  that  after  twenty-four  or  at  most  forty- 
eight  hours,  they  will  be  found  firmly  dried  to  the 
glass.  In  the  process  of  dry  ins;  they  will  have  lost 
a  good  deal  in  thickness,  and  this  is  now  still  further 
reduced  by  planing  off  the  upper  stained  surface  with 
a  razor,  or  other  suitable  instrument,  without  scrap- 
ing away  at  any  place,  if  it  can  be  helped,  the  lower 
stained  part  which  is  adherent  to  the  slide.  The 
further  preparation  consists  in  covering  the  section 
writh  a  thin  layer  of  dammar  varnish  (oil  of  cloves 
is  not  requisite);  after  which  it  may  be  examined 
with  the  microscope,  and,  if  it  appears  satisfactory, 
a  cover- glass  added. 

There  is  hardly  any  limit  to  the  size  of  which  a 
section  of  the  brain  may  be  obtained  by  this  method, 
and,  if  due  care  be  taken  with  the  planing  down  of 
the  section,  it  is  in  nearly  every  case  attended  with 
success.  In  addition  to  exhibiting  nerve-cells,  the 
method  is  useful  for  following  the  course  of  tracts 
of  nerve-fibres  in  the  nervous  centres,  since,  owing 
to  the  depth  of  staining  of  the  axis-cylinders,  the 
nerves  may  be  traced  for  considerable  distances. 

21* 


246  PRACTICAL    HISTOLOGY. 


CHAPTER   XX. 

THE  ORGANS  OF  THE  SENSES — THE  EYE. 
THE  EYELIDS,  SCLEROTIC,  AND  CORNEA. 

THE  study  of  the  eye  should  be  made  as  much  as 
possible  from  that  of  the  human  subject,  for  there 
are  slight  differences  in  the  structure  of  some  of  the 
parts  in  man  and  animals,  and,  moreover,  it  is  on 
the  whole  easier  to  demonstrate  the  structures  in 
the  human  eye.  On  the  other  hand,  there  is  no 
organ  which  it  is  so  absolutely  essential  to  obtain 
in  a  perfectly-fresh  condition.  For  this  and  other 
reasons  it  is  scarcely  possible  to  get  material  from 
the  post-mortem  room,  and  the  only  opportunities 
that  usually  present  themselves  occur  when  an  eye 
is  removed  on  account  of  some  injury  or  disease 
which  is  confined  to  a  particular  part ;  the  other 
intact  portions  may  in  such  cases  be  available  for 
histological  purposes.  In  rare  instances  an  entire 
healthy  eye  has  to  be  removed  (in  operating  for  the 
removal  of  extensive  rodent  ulcers  of  the  brow  and 
face  this  may  be  necessary),  and  if  the  student  should 
be  able  to  procure  such  an  one,  the  following  would 
be  perhaps  the  best  way  to  deal  with  the  excised 
organ  with  the  view  of  making  the  best  use  of  it : 
As  soon  after  removal  as  possible  separate  the  eye, 
by  an  oblique  cut  with  a  very  sharp  knife  or  razor, 
into  two  halves,  an  anterior  and  a  posterior ;  the 
cut  to  start  from  just  behind  the  attachment  of  the 
iris  anteriorly  and  superiorly,  and  pass  downwards 
and  backwards  towards  the  posterior  part  of  the 
organ,  coming  out  a  little  below  the  yellow  spot 
and  optic  nerve.  Then  put  the  posterior  part,  after 


THE    EYE.  247 

allowing  the  vitreous  humor  to  fall  away  from  the 
retina,  into  2  per  cent,  solution  of  osmic  acid,  and 
the  anterior  part  into  Miiller's  fluid.  The  cornea 
is  to  be  cut  through  at  one  place  with  a  sharp  scalpel, 
so  that  the  preservative  fluid  may  get  freely  into 
the  anterior  chamber. 

The  piece  in  osmic  acid  is  left  there  for  eight 
hours ;  it  is  then  placed  in  water  for  two  hours,  and 
finally  transferred  to  a  mixture  of  equal  parts  of 
glycerine,  alcohol,  and  water ;  in  this  it  is  to  remain 
for  a  week  or  more,  until  wanted. 

The  other  piece  is  to  lie  in  Miiller's  fluid  a  fort- 
night, changing  the  fluid  one  or  twice ;  it  is  then 
placed  in  water  for  two  or  three  hours,  then 'in  weak 
spirit  for  a  day  or  two,  and  finally  preserved  in 
strong  spirit.  Of  the  lower  animals,  the  eyes  of  the 
pig  serve  best  for  exhibiting  the  minute  structure, 
especially  of  the  retina.  In  this  animal  the  eye 
corresponds  more  closely  in  point  of  size,  and  ap- 
proaches more  nearly  in  structure  to  the  human  eye 
than  that  of  the  ox  or  sheep,  the  other  animals  the 
eyes  of  which  are  usually  readily  procurable. 

Preparation  1.  The  eyelids. — These  are  studied 
by  making  sections  of  the  hardened  lid  across  its 
long  axis  and  vertically  to  its  surfaces.  The  lid 
may  be  obtained  from  a  still-born  child,  preferably 
one  the  bloodvessels  of  which  have  been  injected. 
It  is  to  be  hardened  in  spirit  and  embedded,  and 
the  sections — which  present  no  unusual  difficulty — 
stained  with  logwood  and  mounted  in  dammar.  In 
this  way  almost  all  the  parts  are  well  displayed ; 
the  skin  with  its  epidermis  on  the  outer  side,  and 
with  a  few  small  hairs  and  sweat  glands  seen  here 
and  there;  the  mucous  membrane  (conjunctiva)  on 
the  inner  side ;  the  Meibomian  glands  cut  along  the 
length  of  their  wide,  straight  duct,  with  their  round 
saccules  lined  with  epithelium  cells  (of  a  whitish 
glistening  appearance,  due  to  the  fatty  secretion 
they  contain,  and  which  also  fills  the  duct);  the 
eyelashes  with  their  large  hair-follicles  and  seba- 


248  PRACTICAL    HISTOLOGY.. 

ceous  glands ;  the  cut  ends  of  the  very  small  muscu- 
lar fibres  of  the  orbicularis  arranged  in  groups,  and 
separated  by  connective  tissue ;  and  the  general  con- 
nective tissue  which  serves  to  unite  all  the  parts 
together,  and,  becoming  denser  towards  the  inner 
surface,  forms  the  so-called  u  tarsal  cartilage,"  long 
described  as  composed  of  fibro-cartilage,  in  reality 
containing  no  cartilage-cells. 

Preparation  2.  The  lachrymal  gland  is  pre- 
pared in  the  same  way  as  the  salivary  glands. 

Preparation  3.  The  substance  of  the  scle- 
rotic.— The  sclerotic  is  studied  by  means  of  sections 
made  from  an  eye  that  has  been  hardened  either  in 
spirit  or  in  chromic  acid  and  spirit,  the  sections 
being  stained  with  logwood.  There  is  nothing  spe- 
cial about  these  preparations,  and  they  present  no 
particular  interest.  But  covering  the  outer  surface 
of  the  globe  is  a  loose  connective  tissue  membrane, 
the  capsule  of  Tenon,  composed  of  two  apposed 
layers,  lined  by  epithelioid  cells  which  bound  a 
lymph  space,  and  covering  the  inner  surface  of  the 
sclerotic  is  another  delicate  lamella  of  loose  connec- 
tive tissue,  closely  adherent  to  the  fibrous  substance 
of  the  coat,  and  of  a  brown  appearance,  due  to  the 
presence  of  pigment.  This  layer  (the  lamina  fusca) 
is  also  bounded  internally  by  a  layer  of  epithelioid 
cells,  and  is  separated  from  a  similar  lamella  (the 
lamina  suprachoroidea)  on  the  outer  surface  of  the 
choroid  coat  by  another  lymph  space,  traversed  here 
and  there  by  the  vessels  and  nerves  as  they  pass  ob- 
liquely across  it  from  the  sclerotic  to  the  choroid. 

Preparation  4.  Capsule  of  Tenon.— To  exhibit 
the  epithelioid  cells  of  the  capsule  of  Tenon  a  fresh 
eye  is  taken,  and  the  adhering  orbital  fat,  and  every- 
thing but  the  insertions  of  the  eye-muscles,  removed 
from  the  globe,  leaving  the  loose  connective  tissue 
membrane.  The  eye  thus  cleared  is  rinsed  in  dis- 
tilled water,  and  a  few  drops  of  nitrate  of  silver 
solution  are  poured  over  the  posterior  part.  After 
three  minutes  the  silver  is  rinsed  off  again  by  a 


THE    EYE.  249 

stream  of  distilled  water,  and  the  eye  is  placed  in 
water  in  the  sunlight.  When  sufficiently  stained  it 
is  removed  from  the  window,  fastened  under  water 
to  a  loaded  cork  by  a  long  pin  passed  through  the 
cornea,  and  a  piece  of  the  capsule  of  Tenon  is  dis- 
sected off  the  globe,  floated  flat  on  to  a  slide,  and 
removed  from  Ithe  fluid.  After  the  excess  of  water 
has  been  got  rid  of,  the  piece  is  covered  in  glycerine 
and  examined  for  the  epithelioid  markings. 

Preparations  5  and  6.  Lamina  fusca.— The 
epithelioid  layer  lining  the  lamina  fusca  is  also  pre- 
pared by  nitrate  of  silver.  A  square  piece  of  the 
sclerotic  is  dissected  off  from  a  fresh  eye  ;  the  convex 
outer  surface  of  the  piece  is  then  pressed  in  and  made 
concave,  the  previously  concave  inner  surface  being 
made  the  convex  one,  and  the  piece  is  first  dipped 
in  water,  then  placed  for  two  minutes  in  silver  solu- 
tion, then,  after  being  again  rinsed  in  water,  trans- 
ferred to  spirit  and  placed  in  the  light,  with  the 
inner  surface  or  lamina  fusca  uppermost.  After  half 
an  hour,  by  which  time  even  in  diffused  daylight 
the  silver  will  probably  be  reduced,  although  owing 
to  the  natural  brown  color  this  cannot  well  be  seen, 
it  is  removed,  and  in  twenty-four  hours,  or  when 
hard  enough  for  the  purpose,  sections  are  made  from 
the  brown  surface,  placed  in  water,  and  mounted  in 
glycerine.  The  pigment  cells  usually  obscure  the 
silver  markings  to  a  certain  extent.  This  inconve- 
nience can  be  obviated  by  using  the  eye  of  an  albino 
rabbit.  Here,  moreover,  the  sclerotic  is  not  too 
thick  to  admit  of  the  piece  being  mounted  entire 
in  glycerine ;  the  immersion  and  hardening  in  spirit 
are  then  not  necessary,  for  the  piece  may  be  exposed 
in  water  to  the  light,  and  mounted  without  further 
preparation,  two  or  three  radial  slits  being  made  in 
it  if  necessary  with  the  object  of  causing  it  to  lie 
flat  on  the  slide. 

Besides  this  preparation  of  its  epithelioid  layer 
the  lamina  fusca  may  itself  be  displayed  in  an  eye, 
or  portion  of  an  eye,  that  has  been  prepared  with 


250  PRACTICAL    HISTOLOGY. 

Miiller's  fluid.  To  obtain  it  a  small  piece  of  the 
sclerotic  is  pinned  to  a  cork  or  wax-cake  under  weak 
spirit  (equal  parts  of  water  and  spirit) ;  and  the 
lamina  fusca  is  dissected  oft'  from  its  inner  surface, 
and  floated  on  to  a  slide,  the  spirit  being  then  al- 
lowed to  evaporate  so  as  to  leave  the  delicate  mem- 
brane moistened  only  with  water.  The  preparation 
may  now  be  covered,  and  glycerine  added  at  the 
edge  of  the  cover-glass. 

Preparation  77  Sections  of  cornea,— The  seve- 
ral layers  of  which  the  cornea  is  composed,  and  their 
relative  thickness,  should  first  be  studied  in  sections 
made  vertically  to  its  surfaces.  For  this  purpose 
the  anterior  part  of  an  eye  (pig's  or  ox's  if  a  human 
eye  is  riot  procurable)  is  placed  in  2  per  cent,  solu- 
tion of  bichromate  of  potash  for  fourteen  days. 
(Miiller's  fluid  may  be  used  instead,  but,  for  the 
cornea,  possesses  no  advantage  over  the  simple  bi- 
chromate, although,  if  the  retina  is  to  be  examined, 
Mailer's  fluid  should  be  employed.)  It  is  as  well  to 
remove  the  lens  so  that  the  solution  passes  freely  to 
the  posterior  surface  of  the  cornea.  After  the  time 
specified  the  tissue  is  put  into  weak  spirit  for  twenty- 
four  hours,  and  then  transferred  to  strong  spirit. 
In  two  or  three  days  more  it  will  be  ready  for  mak- 
ing sections.  For  this  purpose  a  piece  of  the  cornea 
is  cut  out  and  embedded  in  wax-mass.  Very  thin 
sections  vertical  to  the  surface  are  to  be  made, 
stained  in  logwood,  and  mounted  in  dammar.  In 
this  process  a  source  of  difficulty  is  sometimes  met 
with,  in  the  curling  up  of  the  posterior  part  of  the 
section  when  transferred  from  spirit  to  oil  of  cloves, 
after  having  been  stained.  This  can  sometimes  be 
got  rid  of  without  spoiling  the  section,  by  careful 
manipulation  with  needles  ;  but,  if  it  be  found  im- 
possible to  obviate  it  in  any  other  way,  the  plan 
may  be  adopted  of  placing  each  section,  after  it  has 
as  usual  been  stained  with  logwood,  and  rinsed  in 
water,  in  absolute  alcohol  for  a  few  minutes,  trans- 
ferring it  to  a  slide,  and  immediately  covering  it  with 


THE    CORNEA.  251 

the  thin  glass.  Oil  of  cloves  is  then  allowed  to  run 
under  and  clarify  the  specimen,  which  is  prevented 
from  curling  up,  in  consequence  of  the  pressure  of 
the  cover-glass. 

Preparation  8.  Epithelium  of  the  cornea, — 
The  stratified  epithelium  covering  the  front  of  the 
cornea  is  well  seen  in  the  vertical  sections,  but  the 
characters  of  the  individual  cells  which  compose  it 
must  be  studied  in  a  teased  preparation.  For  this 
purpose  a  piece  of  the  cornea  is  placed  in  a  compara- 
tively large  quantity  of  J-  per  cent,  bichromate  of 
potash  solution,  and  allowed  to  remain  in  this  for  a 
week,  changing  the  fluid  once  or  twice  during  that 
time.  Then  with  the  point  of  a  scalpel,  or  spear- 
headed needle,  a  small  portion,  including  however 
the  whole  thickness  of  the  epithelium,  is  scraped  off 
the  front,  placed  in  a  drop  of  distilled  water  on  a 
slide,  and  broken  up  with  needles  as  finely  as  pos- 
sible. A  piece  of  hair  is  added,  and  lastly  the 
cover-glass,  and  the  specimen  is  then  ready  for  ex- 
amination. The  cells  of  the  various  layers  will  be 
recognized  by  the  characteristic  forms  they  present ; 
those  of  the  deepest  layer  being  in  shape  like  a  rifle- 
bullet,  those  next  above  cupped  to  receive  the 
rounded  or  conical  ends  of  the  deeper  cells,  and  the 
superficial  layers  being  more  flattened  as  they  are 
nearer  the  surface.  The  fine  ridges  and  furrows  on 
many  of  the  cells  can  be  distinctly  made  out  with 
a  high  power,  and  give  a  jagged  contour  to  the  cell. 

To  preserve  the  preparation  permanently  in  glyce- 
rine it  should  first  be  stained  with  logwood.  This 
is  readily  done  by  applying  a  drop  of  a  very  weak 
solution  to  the  edge,  and  allowing  it  to  diffuse  under 
the  cover-glass;  after  a  short  time  glycerine  is  added 
at  the  same  edge,  and  gradually  replaces  the  log- 
wood solution,  the  water  from  which  evaporates 
meanwhile  at  the  other  borders  of  the  cover-glass. 

Preparation  9.  The  substantia  propria  of 
the  cornea, — The  fibrous  structure  of  the  sub- 
stantia propria  of  the  cornea  can  readily  be  seen  by 


252  PRACTICAL    HISTOLOGY. 

teasing  out  either  a  fresh  cornea,  or  one  which  has 
been  macerated  for  a  while  in  weak  bichromate  of 
potash,  or  in  picric  acid.  The  lamellse  which  the 
fibrous  bundles  form  are  apparent  when  an  attempt 
is  made  to  tear  the  corneal  tissue,  and  they  are  well 
seen,  cut  in  different  directions,  in  the  vertical  sec- 
tions previously  made. 

The  corneal  corpuscles  are  visible  in  the  sections 
as  mere  lines,  each  with  an  enlargement  in  the 
middle,  stretching  across  the  containing  cell-space, 
which  is  fusiform  in  section,  and  is  seldom  filled  by 
the  included  corpuscle.  These  appearances  are  best 
observed  in  the  human  cornea,  but  may  also  be 
made  out  in  that  of  the  pig  and  those  of  other  ani- 
mals. But  although  they  look  like  mere  lines  in 
vertical  section,  they  are,  like  most  other  connective 
tissue  cells,  in  reality  flattened  out  conformably  to 
the  surfaces  of  the  lamellae,  and  present  when  viewed 
flat  great  irregularities  of  form,  and  numerous 
branching  processes  with  which  they  come  in  many 
cases  into  connection  with  one  another.  They  are 
best  brought  to  view  by  the  gold  method,  and, 
since  this  also  serves  to  show  the  nerves,  the  two 
structures  may  be  studied  in  the  same  preparation. 

Preparation  10.  Corpuscles  and  nerves  of 
the  frog's  cornea. — The  brain  and  spinal  cord  of 
a  frog  having  been  destroyed,  the  animal  is  laid  on 
the  table  or  held  by  an  assistant,  and  the  membrana 
nictitans  of  the  eye  is  seized  with  forceps,  and  en- 
tirely removed  by  two  or  three  snips  with  fine, 
sharp-pointed  scissors.  The  animal  is  then  taken 
up  and  held  in  the  operator's  left  hand,  the  thumb 
pressing  upwards  under  the  lower  jaw,  so  that  the 
eyes  are  made  as  prominent  as  possible,  and  the 
point  of  one  of  the  scissor  blades  is  inserted  into  the 
globe  of  the  eye,  just  behind  the  insertion  of  the 
glistening,  yellowish  iris.  By  a  series  of  snips  made 
round  the  eyeball  at  this  plane,  the  anterior  part, 
with  the  cornea,  iris,  and  lens,  is  severed  from  the 
posterior,  and  removed  to  a  watch-glass  containing 


THE    CORNEA.  253 

salt  solution.  Then  whilst  the  edge  of  the  cut  scle- 
rotic is  held  by  the  one  pair  of  forceps,  with  another 
pair  the  iris  is  seized  close  to  the  same  spot,  and  is 
easily  torn  away  from  the  sclerotic,  bringing  the  lens 
with  it.  So  that  only  the  cornea,  together  with  a 
narrow  rim  of  sclerotic,  is  now  left,  and  since  it  is 
floating  in  fluid  it  retains  its  convexo-concave  form, 
and  all  crumpling  of  the  tissue  is  avoided.  The  salt 
solution  is  now  poured  ofl',  leaving,  however,  just 
enough  to  float  the  cornea  in,  arid  the  watch-glass  is 
filled  up  with  one-half  per  cent,  of  chloride  of  gold 
solution.  The  cornea  is  allowed  to  remain  in  this  a 
full  hour;  it  is  then  removed  to  a  beaker  of  water 
acidulated  with  acetic  acid,  and  is  placed  in  a  warm 
place  in  the  sunlight  (see  p.  96).  After  two  days 
the  fluid  in  the  beaker  is  renewed,  a  teaspoonful  of 
methylated  spirit  being  added  to  prevent  the  growth 
of  fungi,  and  in  two  days  more  the  cornea  may  be 
taken  out  and  prepared  for  the  microscope.  It  is 
first  placed  in  a  flat  dish  of  distilled  water,  and  the 
epithelium,  which  is  very  dark  and  opaque,  is  gently 
scraped  off  the  anterior  surface.  This  done,  the 
sclerotic  rirn  is  cut  ofl'  with  scissors.  It  is  as  well 
to  change  the  wrater  at  this  stage,  so  as  to  get  rid  of 
the  debris  of  epithelium.  The  next  process  consists 
in  the  separation  of  the  corneal  substance  into  two, 
three,  or  more  thin  lamellae.  With  a  little  practice 
it  is  not  at  all  difficult,  thin  as  the  object  already 
seems,  by  holding  it  down  at  one  edge  with  a  pair 
of  forceps  and  working  from  the  same  edge  with  an- 
other pair,  to  separate  a  very  thin  lamina  from  the 
concave  posterior  surface,  consisting  of  the  membrane 
of  Descemet  and  a  delicate  layer  of  the  proper  sub- 
stance of  the  cornea  with  its  corpuscles.  This  poste- 
rior lamella  is  not  only  the  easiest  to  obtain,  but  is 
also,  in  the  frog's  cornea,  the  most  important,  for  it 
contains  the  closest  and  finest  plexus  of  nerves.  To 
mount  it,  all  that  is  necessary  is  to  float  it  on  to  a 
glass  slide,  to  cover  the  preparation,  and  add  glyce- 
rine at  the  ed«;e  of  the  cover-glass.  But.  since  the 
99 


254  PRACTICAL    HISTOLOGY. 

membranous  layer  thus  obtained  has  naturally  the 
convex  shape  of  the  cornea,  and  it  is  of  course  de- 
sirable that  it  should  lie  flat  upon  the  slide  with- 
out creases,  it  is  well  before  mounting  to 
make  three  or  four  radical  snips  in  it  in 
the  way  shown  in  the  adjoining  cut ;  these 
will  enable  it  to  flatten  out  without  fold- 
ing, when  placed  on  the  slide  and  covered. 
Moreover  it  is  important  to  examine  the 
object  with  a  low  power  previously  to  covering  it, 
so  that  any  folds  of  the  membrane,  or  any  foreign 
matter  or  remains  of  epithelium  adhering  to  it,  may 
be  detected  and  removed. 

The  remaining  anterior  part  of  cornea  may  be 
further  separated  in  the  same  way  into  lamellae, 
which  are  to  be  mounted  with  the  same  precautions 
as  the  posterior  lamella.  It  is  not  always  easy  to 
get  them  in  quite  so  complete  a  layer,  but  for  most 
purposes  a  small  shred  will,  if  equally  thin,  show 
nearly  as  much  as  an  entire  lamella. 

In  all  these  specimens  the  corneal  corpuscles  are 
stained  of  a  violet  color,  varying  in  tint  according 
to  the  success  of  the  preparation,  their  nuclei  being 
left  nearly  unstained.  The  nerves  are  colored  almost 
black,  the  fibrils  looking  like  fine  wires  running 
singly  and  in  bundles,  and  provided  along  their 
course  with  numerous  minute  varicosities. 

Preparation  11.  The  corpuscles  and  nerves 
of  the  rabbit's  cornea. — The  cornea  of  the  rabbit, 
or  of  any  other  mammal  recently  killed,  is  prepared 
with  chloride  of  gold  in  the  same  way  as  that  of 
the  frog.  The  eyelids  are  first  removed,  care  being 
taken  in  doing  this  not  to  get  the  hair  on  to  the 
surface  of  the  cornea.  The  eye  is  then  to  be  made 
prominent ;  either  by  an  assistant  who  holds  it 
firmly  with  forceps  thrust  back  in  the  orbit,  so  as 
to  seize  one  or  other  of  the  eye  muscles  near  their 
attachment ;  or  without  an  assistant  by  clamp-for- 
ceps, which  are  inserted  in  like  manner,  and  by  their 
weight  force  the  eyeball  forward  without  unduly 


THE    CORNEA.  255 

compressing  it.  Then  the  cornea,  iris,  and  lens  are 
removed  together,  after  cutting  round  the  sclerotic 
with  scissors,  and  are  placed  in  salt  solution,  and 
finally  the  iris  and  lens  are  removed  in  the  same  way 
as  in  the  preceding  preparation,  and  the  cornea  is 
immersed  in  gold  solution.  Since  it  is  much  thicker 
than  the  frog's  cornea,  it  should  remain  in  the  chlo- 
ride of  gold — which  does  not  penetrate  very  rapidly 
— a  full  hour  and  a  half,  after  which  it  is  placed  in 
acidulated  water  in  the  light,  and  otherwise  treated 
in  the  same  way  as  the  frog  cornea.  But  as  it  is 
not  so  easy  to  separate  the  mammalian  cornea  into 
lamellae,  it  is  better  after  four  days  to  place  the 
stained  cornea  in  spirit  for  twenty-four  hours,  when 
thin  sections  parallel  to  the  surface  may  be  cut,  and 
mounted  in  glycerine.  Embedding  in  wax-mass  is 
not  necessary  if  the  cornea  is  held  in  the  left  hand, 
and  allowed  to  rest  over  the  end  of  the  forefinger, 
the  razor  being  directed  from  the  operator. 

Preparation  12.  Isolation  of  corneal  cor- 
puscles.— After  the  corneal  corpuscles  and  nerves 
have  been  stained  with  gold  in  this  way,  they  can 
be  isolated  by  dissolving  away  the  intermediate 
substance  by  caustic  alkali,  the  action  being  arrested 
before  the  corpuscles  and  nerves,  which  are  more 
resisting  than  the  connective  tissue  bundles,  are 
are  also  destroyed.  With  this  object  a  part  or  the 
whole  of  a  gold-stained  cornea — whether  of  frog  or 
mammal — is  placed,  divested  of  epithelium,  in  a 
watch-glass  containing  a  strong  (20  per  cent.)  solu- 
tion of  caustic  potash  or  soda,  and  this  is  then  put 
into  a  warm  chamber  at  40°  C.  At  the  expiration 
of  three-quarters  of  an  hour  the  tissue,  which  is  now 
quite  soft  and  pulpy,  is  removed  with  a  section  lifter, 
and  placed  in  a  vessel  containing  a  large  quantity 
of  water  faintly  acidulated  with  acetic  acid.  Small 
portions  may  then  be  taken  up  and  mounted,  with 
or  without  further  breaking  up,  in  glycerine.  The 
corpuscles  are  beautifully  displayed,  forming  a  con- 


256  PRACTICAL    HISTOLOGY. 

tinuous  network  by  the  junction  of  their  branches  ; 
and  nervous  fibrils  may  be  seen  intercalated  amongst 
the  corpuscles,  but  never  actually  joined  to,  or  con- 
tinuous with,  the  cells. 

Preparation  13.  Nerves  of  the  rabbit's  cor- 
nea.— For  exhibiting  the  nerves  of  the  cornea  with- 
out at  the  same  time  staining  the  corpuscles  and 
the  epithelium,  the  following  modification  of  the 
gold  method  may  be  used  (Klein).  The  cornea  of  a 
rabbit  or  guinea-pig  is  put  into  half  per  cent,  solu- 
tion of  chloride  of  gold  for  an  hour  and  a  half.  It 
is  removed  from  the  gold  into  distilled  water  and 
placed  in  the  light  (without  warmth)  for  from 
twenty-four  to  thirty  hours,  or  until  the  larger 
nerve  trunks  begin  to  be  visible  near  the  circum- 
ference, converging  towards  the  centre  as  irregular, 
branching  lines.  When  the  staining  has  arrived 
at  this  stage,  and  before  the  corneal  substance  gene- 
rally begins  to  acquire  a  violet  appearance,  the 
cornea  is  removed  from  -the  water  and  placed  in  a 
mixture  of  glycerine  and  water  (one  part  glycerine 
to  two  parts  distilled  water).  After  it  has  been  in 
this  for  twenty-four  hours,  or  longer,  the  corneal 
substance  should  be  very  little  darker  than  before, 
but  the  nerves  much  more  distinct,  and  on  holding 
the  cornea  between  the  finger  and  thumb,  and  mak- 
ing sections  from  the  anterior  surface,  including  the 
epithelium,  and  a  little  of  the  substantia  propria, 
these,  when  covered  in  the  glycerine  mixture,  will 
show  not  only  the  fine  and  close  plexus  of  nerves 
which  lies  immediately  underneath  the  epithelium, 
but  also  the  far  more  minute  network  of  varicose 
ultimate  tibrils  which  extends  between  the  epithe- 
lium cells  almost  to  the  anterior  surface  of  the  epi- 
thelium. If  not  at  first  sufficiently  evident,  these 
intra-epithelial  nerves  may  generally  be  brought 
more  clearly  into  view  by  placing  a  section  for  a 
few  minutes  in  the  strong  caustic  potash  solution. 
From  this  it  is  transferred  by  a  section  lifter  to 


THE    CORNEA.  257 

water,  floated  on  to  a  slide,  removed  from  the  water, 
and  covered,  glycerine  being  afterwards  added  at 
the  edge  of  the  cover-glass.  To  prevent  the  latter 
from  pressing  on  the  softened  tissue,  two  narrow 
slips  of  thin  glass,  which  may  be  cut  with  a  writing 
diamond,  are  to  be  placed  one  on  either  side,  before 
placing  the  cover-glass  over. 

Preparation  14  and  15.  The  cell-spaces  of 
the  cornea. — These  are  shown  in  two  ways  ;  by  the 
silver  method,  and  by  the  method  of  puncture  injec- 
tion. 

The  demonstration  of  the  cell-spaces  by  the  silver 
method  may  be  attempted  in  the  cornea  of  the  frog. 
The  animal  is  decapitated  and  the  brain  destroyed. 
The  eyelid  is  then  removed,  and  the  eye  having  been 
made  prominent  by  the  pressure  of  the  thumb  in 
the  manner  previously  recommended,  the  epithelium 
is  scraped  off  the  front  of  the  cornea  with  a  sharp 
scalpel.  The  cornea  is  then  rubbed  with  a  stick  of 
fused  nitrate  of  silver  (lunar  caustic).  After  live 
minutes  the  surface  is  thoroughly  washed  with  a 
stream  of  distilled  water  from  a  wash-bottle.  The 
head  is  now  placed  in  spirit  in  the  light ;  in  a  short 
time  (from  a  few  minutes  to  an  hour),  when  the 
cornea  is  browned,  the  vessel  containing  it  is  re- 
moved from  the  window  and  left  in  a  dark  place  for 
twenty-four  hours.  The  cornea  is  then  sliced  off, 
placed  in  water,  where  any  remaining  patches  of 
epithelium  are  now  removed,  slit  in  a  triradiate 
manner,  so  that  it  may  lie  flat  on  a  slide,  and  finally 
mounted  in  glycerine. 

The  cornea  of  mammals  may  be  prepared  with 
silver  in  a  similar  manner,  but,  being  generally 
thicker  than  in  the  frog,  it  is  necessary  to  allow  the 
caustic  a  longer  time  to  penetrate,  and,  in  the  final 
preparation,  to  prepare  sections  parallel  to  the 
surface,  instead  of  mounting  the  cornea  whole. 

To  inject  the  cell-spaces  of  the  cornea  by  the  punc- 
ture method,  the  eye  of  the  pig  or  guinea-pig  may 

be  used,  if  a  human  eve  is  not  procurable  for  the 

go* 


258  PRACTICAL    HISTOLOGY. 

purpose.  A  solution  of  alkanet  in  turpentine  is  the 
fluid  which  should  be  chosen,  and  the  mercurial 
pressure  apparatus  (Fig.  30)  is  used.  The  tube  and 
fine  steel  canula  having  been  filled  with  the  alkanet 
solution  to  the  exclusion  of  air-bubbles,  the  canula  is 
inserted  obliquely  into  the  substance  of  the  cornea, 
without  allowing  the  point  to  pass  through  into  the 
anterior  chamber.  The  pressure  is  then  gradually 
raised  to  about  two  inches  of  mercury,  when  the  red 
fluid  should  begin  gradually  to  fill  the  cell-spaces, 
and  to  spread  through  them  over  a  considerable  part 
of  the  cornea.  Indeed,  if  the  injection  is  long  enough 
continued,  the  fluid  may  extend  itself  even  beyond 
the  cornea  1  margin,  and  may  penetrate  into  the  cell- 
spaces  in  the  anterior  part  of  the  sclerotic  coat.  The 
operation  may  with  care  be  successfully  performed 
without  the  mercurial  apparatus,  using  merely  a 
Pravaz  syringe.  But  it  is  difficult  to  avoid  the  pro- 
duction of  extravasation  near  the  point  of  the  canula. 
This  does  not,  however,  always  militate  against  the 
success  of  the  experiment,  for  beyond  the  limit  of 
the  extravasation  the  fluid  may  slowly  penetrate 
into  the  cell-spaces  of  the  tissue,  and  this  may  go 
on  even  after  the  syringe  has  been  removed,  espe- 
cially if  the  cornea  is  cut  out,  laid  flat  on  a  slide, 
and  allowed  to  dry.  As  watery  fluid  becomes  with- 
drawn from  the  cell-spaces  in  the  process  of  drying, 
the  alkanet  solution  tends  to  pass  in  to  occupy  its 
place. 

Another  very  successful  plan  of  inducing  the  in- 
jecting fluid  to  pass  from  such  an  extravasation  into 
the  neighboring  cell-spaces  consists  in  gently  stroking 
from  the  extravasation  towards  the  margin  of  the 
cornea  with  a  smooth  instrument,  such  as  a  glass 
rod  or  the  ivory  handle  of  a  scalpel.  But  if  too 
great  pressure  is  exerted  upon  the  fluid  it  will  be 
found  that,  in  place  of  taking  the  closely  reticulating 
course  which  it  would  pursue  if  it  merely  occupied 
the  cell-spaces,  the  injection  tends  to  shoot  through 
the  tissue  in  straight  lines,  which  in  successive  planes 


THE    COKNEA.  259 

of  the  cornea!  tissue  run  at  right  angles  to  one  an- 
other. These  lines  represent  the  "  corneal  tubes"  of 
Bowman.  Their  appearance  is  due  to  the  fact  that 
the  pressure  exerted  has  been  sufficient  to  force  the 
injecting  fluid  into  the  interstices  between  the  con- 
nective tissue  bundles,  pushing  these  asunder,  and 
burrowing  its  way  through  the  soft  ground  substance 
which  unites  the  bundles  and  lamellae.  And,  since 
the  cell-spaces  occur  in  this  ground  substance,  the 
existence  of  a  slight  enlargement  or  fusiform  swelling 
here  and  there  on  the  tubes  is  accounted  for. 

These  corneal  tubes  then  are  to  be  looked  upon  as 
purely  artificial  products,  not  corresponding  with 
any  pre-existing  channels  in  the  tissue  (except  per- 
haps when  the  fluid  passes  along  the  sheath  of  a 
nerve).  They  are  always  obtained  when  any  fluid 
which  is  not  able  to  penetrate  into  the  cell-spaces  is 
forced  into  the  substance  of  the  cornea.  They  are 
seen  when  mercury  is  injected  by  the  puncture 
method,  and  this  is  how  they  were  first  obtained  by 
Bowman;  and  if  air  be  forced  with  a  syringe  into 
the  tissue  a  similar  effect  is  produced.  In  all  cases 
the  tubes  cease  abruptly  at  the  corneo-sclerotic  junc- 
tion, where  the  connective  tissue  becomes  denser  and 
has  a  less  regular  arrangement. 

Preparation  16.  Parts  at  the  junction  of 
the  cornea  with  the  sclerotic. — The  corneo-scle- 
rotic  junction,  the  ciliary  muscles,  and  the  iris  are 
all  well  seen  in  their  relations  to  one  another  in  a 
meridional  section  of  the  part  of  the  eye  where  they 
are  situated.  The  section  may  be  made  from  the 
anterior  segment  which  we  have  assumed  to  have 
been  hardened  in  M tiller's  fluid  and  in  spirit  (p.  247). 
It  is  not  necessary  to  embed  this  entire,  but  sufficient 
to  cut  out  with  sharp  scissors  under  spirit  a  piece 
which  includes  all  the  parts  above  enumerated.  The 
piece  is  first  placed  in  oil  of  cloves  for  a  few  minutes; 
it  is  then  transferred  to  cacao-butter,  which  is  kept 
just  melted  over  a  sand-bath,  and  is  allowed  to  re- 
main in  this  for  fully  two  hours,  so  that  the  butter 


260  PRACTICAL    HISTOLOGY. 

may  soak  into  every  part  of  the  tissue.  Meanwhile 
a  paper  mould  of  the  usual  form  is  filled  with  the 
melted  cacao-butter,  which  is  allowed  to  become 
solid,  and  in  the  cake  thus  produced  an  excavation 
is  made  near  one  end  large  enough  to  receive  the 
piece  to  be  embedded  which  is  placed  in  the  hollow 
in  such  a  position  that  radial  sections  vertical  to  the 
surface  may  be  made,  and  the  excavation  is  then 
filled  up  with  melted  cacao-butter.  This  in  harden- 
ing sets  into  one  piece  with  the  rest  of  the  cake,  and 
in  cutting  the  sections  the  advantage  is  gained  that 
all  the  parts  have  exactly  the  same  density  and  offer 
a  uniform  resistance  to  the  razor.  Much  thinner 
sections  may  therefore  be  made  by  this  method  than 
by  any  other.  The  sections,  as  they  are  obtained, 
are  placed  in  oil  of  cloves,  which  slowly  dissolves  the 
cacao-butter  out  from  the  tissue ;  the  process  can  be 
accelerated  by  slight  warmth.  They  are  next  placed 
in  alcohol  to  remove  the  oil  of  cloves ;  then  for  a  few 
minutes  in  Kleinenberg's  logwood;  and  are  finally 
passed  again  through  alcohol  and  oil  of  cloves,  to  be 
mounted  in  dammar  varnish  in  the  usual  way.  Few 
specimens  better  repay  the  trouble  of  preparation 
than  these.  The  cornea,  sclerotic,  iris,  choroid,  liga- 
menturn  pectinatum,  canal  of  Schlemm,  ciliary  mus- 
cle, both  radial  and  circular,  and  even  the  ora  serrata 
and  pars  ciliaris  of  the  retina  are  all  exhibited  with 
the  greatest  clearness  in  a  successful  section,  and 
their  structure  arid  relations  may  be  advantageously 
studied. 

THE  CHOROID  AND  IRFS. 

The  choroid  coat  is  to  be  prepared  from  an  eye 
hardened  in  Miiller's  fluid.  Besides  the  main  sub- 
stance of  the  coat  containing  the  larger  bloodvessels, 
the  lamina  suprachoroidea,  thechorio-capillaris,  and 
the  membrane  of  Bruch,  should  all  be  separately 
displayed.  The  posterior  attachment  of  the  fibres 
of  the  ciliary  muscle,  and  the  gangliated  plexus  of 


THE    CILIARY    MUSCLE.  261 

nerves  which  is  found  in  the  neighborhood  of  its 
posterior  attachment,  will  be  exhibited  with  the 
lamina  suprachoroidea. 

Preparation  17.  Ciliary  muscle  and  lamina 
suprachoroidea, — To  prepare  these,  the  anterior 
part  of  the  eye  is  to  be  pinned  down  under  spirit, 
and  the  cornea  and  sclerotic  cut  away  at  one  part, 
when  the  radiating  fibres  of  the  ciliary  muscle  will 
be  seen  passing  meridionally  from  their  origin 
opposite  the  attachment  of  the  iris,  and  forming  a 
layer  which  becomes  gradually  thinner  as  it  extends 
backwards  and  finally  ceases  in  the  superficial  part 
of  the  choroid.  A  small  piece  of  the  muscle  is 
seized  with  sharp  forceps  near  its  origin,  and,  by 
carrying  the  instrument  slowly  backwards,  is  gradu- 
ally torn  away  from  the  rest.  It  will  be  found  that 
the  shred  which  comes  away  generally  spreads  out 
posteriorly  into  a  very  thin  membranous  lamina, 
this  being  in  fact  a  piece  of  the  lamina  suprachoroi- 
dea into  which  the  superficial  fibres  of  the  ciliary 
muscle  are  inserted.  A  considerable  length  may  be 
torn  off  in  this  way,  and  the  piece  so  obtained  is  to 
be  floated  directly  on  to  a  slide,  Avhich  is  dipped  for 
the  purpose  into  the  spirit.  It  must  be  moved  with 
great  care,  so  as  to  avoid  folds  or  creases.  The  slide 
is  quickly  wiped  free  of  spirit,  with  the  exception  of 
that  which  immediately  moistens  the  specimen,  and 
a  drop  of  freshly-filtered  logwood  placed  upon  the 
tissue,  and  allowed  to  remain  on  it  for  ten  minutes. 
The  staining  solution  is  then  poured  off,,  and  the 
remains  of  it  are  removed  by  allowing  a  drop  or  two 
of  water  to  flow  gently  over,  without  disturbing  the 
position  of  the  membrane.  Finally  a  cover-glass  is 
laid  on,  and  a  drop  of  glycerine  allowed  to  run  in  at 
the  edge  of  the  cover-glass. 

The  preparation  so  obtained  is,  if  successful,  a 
very  striking  one.  Besides  the  branched  pigment 
cells  of  the  choroidal  tissue,  and  a  certain  number  of 
cells,  similar  to  white  blood  corpuscles,  911  the  sur- 
face of  the  membrane,  a  number  of  large,  round  or 


262  PRACTICAL    HISTOLOGY. 

oval  nuclei  are  seen  in  the  lamina,  which  are  appa- 
rently devoid  of  cell-body.  These  are  in  reality  the 
nuclei  of  epithelioid  cells  which  bound  the  lamina 
suprachoroidea  externally,  and  serve  as  part  of  the 
lining  of  the  lymphatic  space  which  lies  between 
this  and  the  lamina  fusca  of  the  sclerotic.  Their 
outlines  cannot  be  brought  to  view  without  the  aid 
of  nitrate  of  silver,  and  the  cell-bodies  are  too  deli- 
cate and  transparent  to  be  shown  by  the  present 
mode  of  preparations.  A  large  number  of  elastic 
fibres  are  also  seen  on  the  membrane,  especially  at 
the  terminations  of  the  fibres  of  the  ciliary  muscle, 
where  they  appear  to  come  in  relation  with  the  ends 
of  these,  an  elastic  fibre  passing  for  a  certain  dis- 
tance along  each  side  of  the  muscular  fibre,  and 
seeming  to  serve  in  this  way  for  its  attachment. 
The  involuntary  fibres  are  particularly  well  shown, 
their  nuclei  being  conspicuously  stained  by  the  log- 
wood ;  many  of  the  bundles  terminate  in  peculiar 
tufts,  from  which  the  fibres  radiate  in  all  directions. 
It  may  happen  that  the  preparation  includes  one  of 
the  long  ciliary  nerves ;  this,  as  it  coursed  forward 
to  enter  the  ciliary  muscle,  having  been  torn  away 
with  the  shred  of  membrane.  If  so,  it  may  be  fol- 
lowed with  the  microscope  and  its  branches  traced 
amongst  the  bundles  of  muscular  fibres,  forming  a 
plexus  with  those  of  the  other  nerves.  In  tracing 
the  branches  characteristic  ganglion-cell  enlarge- 
ments will  here  and  there  be  found  interpolated  in 
the  course  of  a  nerve  fibre. 

Preparation  18.  Vascular  layers  of  choroid 
and  membrane  of  Bruch. — As  seen  in  the  prepara- 
tion just  described,  it  is  easy  to  detach  the  lamina 
suprachoroidea  from  the  rest  of  the  choroid.  The 
other  three  parts  are  more  difficult  to  separate,  and 
their  complete  isolation  may  require  considerable 
time  and  patience.  But  for  demonstrating  their 
structure  it  is  not  absolutely  necessary  for  them  to 
!><•  completely  separated  as  distinct  membranes  ;  it  is 
sufficient  if,  in  a  piece  which  includes  all,  one  or 


THE    IRIS.  263 

other  is  left  projecting  at  the  edge,  so  as  in  this  way 
to  be  seen  distinct  from  the  other  layers.  But  before 
commencing  the  attempt  at  separation,  the  hexago- 
nal pigment  cells,  which  belong  to  the  retina  but 
frequently  adhere  to  the  inner  surface  of  the  choroid, 
must  be  entirely  removed  by  gently  brushing  that 
surface  with  a  hair  pencil.  The  separation  and 
brushing  are  performed  under  fluid  (spirit),  and  will 
be  much  facilitated  by  the  use  of  a  dissecting  lens. 

Preparation  19.  The  musculature  of  the 
iris. — The  circular  and  radiating  plain  muscular 
fibres  of  the  iris  may  be  demonstrated  in  the  albino 
rabbit.  The  eye  is  cut  in  half  and  the  anterior  part 
placed  in  spirit  for  a  day  or  more.  Then  the  lens  is 
removed,  and  the  segment  of  the  iris — including  its 
whole  width,  from  the  pupillary  aperture  to  the 
ciliary  processes  of  the  choroid — is  cut  out  and 
placed  in  dilute  logwood.  When  sufficiently  but 
not  too  deeply  stained,  it  is  put  into  water  for  a 
minute  or  two  to  remove  the  excess  of  staining  fluid, 
then  passed  through  spirit  and  oil  of  cloves,  and 
finally  mounted  in  dammar,  with  the  posterior  sur- 
face uppermost.  The  thick  ring  of  the  sphincter  is 
easily  seen  in  these  preparations,  and  also  the  inter- 
lacing bundles  of  plain  muscular  fibres  of  which  tjrie 
dilatator  is  composed  ;  they  may  be  observed  to  bend 
round  near  the  pupil,  and  take  the  direction  of  and 
blend  with  those  of  the  sphincter.  At  the  circum- 
ference of  the  iris  also,  a  similar  bending  round  of 
the  radiating  fibres  is  observed. 

Preparation  20.  Human  iris. — Although,  in 
consequence  of  the  presence  of  the  uveal  pigment, 
more  troublesome  to  prepare,  it  is  nevertheless  desir- 
able to  make  a  similar  preparation  of  the  human 
iris,  for  the  musculature  is  somewhat  different,  the 
dilatator  forming  a  uniform  thin  expansion,  which 
covers  the  posterior  surface  immediately  under  the 
pigmented  epithelium,  and  not  distinct  radially  ar- 
ranged interlacing  bundles  with  intervening  meshes 
as  in  the  rabbit.  The  specimen  may  be  made  from 


264  PRACTICAL    HISTOLOGY. 

an  eye  that  has  been  in  Miiller's  fluid  or  bichromate 
of  potash  (2  per  cent.),  and  subsequently  in  spirit. 
A  piece  is  cut  out  as  before,  and  is  treated  in  a  simi- 
lar way,  except  that  before  being  stained  the  pigment 
is  brushed  completely  off  the  posterior  surface  with 
a  stiff  camel-hair  brush.  This  must  be  done  under 
spirit,  and  of  course  very  carefully  so  as  to  avoid 
tearing  the  tissue;  during  the  operation  the  iris  is 
examined  now  and  then  with  a  low  power,  to  deter- 
mine when  all  the  pigment  is  removed.  It  is  diffi- 
cult to  prevent  some  of  the  pigment  granules  from 
still  sticking  to  the  surface,  but,  as  they  tend  for  the 
most  part  to  adhere  along  the  lines  of  junction  of 
the  fibre  cells,  their  presence  does  not  spoil  the  object 
of  the  preparation,  for  the  dilatator  fibres  are  if  any- 
thing better  displayed. 

THE  RETINA. 

Preparations  21  and  22.  Sections  of  the 
retina. — It  will  be  well  to  study  the  general  ar- 
rangement of  the  several  layers  of  the  retina  by  means 
of  sections,  before  its  constituent  elements  are  ob- 
served isolated.  Two  methods  of  hardening  may  be 
recommended,  viz.  by  Miiller's  fluid  and  by  osmic 
acid,  the  process  being  in  both  instances  completed 
by  spirit.  If  possible  to  obtain  it  perfectly  fresh  and 
healthy,  the  human  retina  should  always  be  used  ; 
if  not  so  obtainable,  that  of  the  pig  is  preferable  to 
the  retina  of  most  other  animals,  and  in  the  follow- 
ing preparations  it  will  be  assumed  that  the  eyes  of 
that  animal  are  employed. 

To  harden  the  retina  in  Miiller's  fluid  it  is  better 
to  keep  the  bulb  whole,  so  that  the  membrane  re- 
mains supported  by  the  vitreous  humor.  But  to  let 
the  fluid  in  readily  two  or  three  cuts  must  be  made 
in  the  sclerotic  with  a  razor,  or  a  piece  even  be  cut 
out  at  one  or  two  places.  The  eye  is  then  dropped 
into  a  relatively  large  quantity  of  Miiller's  fluid,  and 
allowed  to  remain  in  it  for  a  week,  the  fluid  being 


THE    RETINA.  265 

stirred  at  intervals  ;  the  globe  is  then  cut  across,  and 
left  for  another  week  in  the  liquid,  after  which  time 
it  is  transferred  first  for  twenty-four  hours  to  weak, 
and  then  to  strong  spirit.  In  another  day  or  two 
it  is  ready  for  the  preparation  of  sections.  Small 
pieces  from  different  regions  may  be  taken,  but  as 
they  are  all  treated  in  the  same  way,  they  may  be 
described  as  one: — 

The  staining  of  the  tissue  is  first  effected  by 
placing  the  piece  for  twenty-four  hours  in  alcoholic 
logwood  (Kleinenberg's).  From  this  it  is  transferred 
to  spirit,  then  embedded  in  wax-mass.  It  is  better 
so  to  place  it  in  the  embedding  tray  that  the  sections 
shall  be  both  vertical  and  meridional ;  since,  made 
in  this  way,  they  will  take  the  general  course  of  the 
fibres  of  the  optic  nerve.  The  sections  cannot  be 
made  too  thin,  but  they  should  be  complete,  that  is 
to  say,  they  should  include  all  the  layers  of  the 
membrane.  One  or  two  of  the  thinnest  are  to  be 
mounted  in  glycerine  ;  the  others  may  be  transferred 
from  the  spirit  to  oil  of  cloves,  and  mounted  in 
dammar.  In  these  preparations  the  nerve  fibres, 
the  Mullerian  fibres,  the  inner  and  outer  granules, 
and  in  fact  the  layers  generally,  are  all  well  dis- 
played, but  the  rods  arid  cones  are  much  altered,  and 
the  natural  form  and  appearance  .of  the  other  elements 
is  by  no  means  well  preserved. 

In  this  respect  the  osmic  method  yields  far  better 
results.  The  bulb  in  this  case  is  cut  open,  and  either 
the  whole  or  a  piece  only  of  the  retina  is  taken  arid 
placed  in  2  per  cent,  solution  of  osrnic  acid.  Here 
it  is  left  for  several  hours,  and  then,  after  being  an 
hour  or  two  in  water  to  get  rid  of  the  excess  of 
osmic  acid,  it  is  transferred  to  alcoholic  logwood  for 
twenty -four  hours.  From  this  it  is  placed  in  strong 
spirit,  where  it  may  be  left  until  wanted.  It  is  well 
to  embed  the  piece  by  the  cacao-butter  process,  since 
by  hardly  any  other  method  can  sufficiently  thin 
and  complete  sections  of  a  retina  that  has  been 
hardened  in  osmic  acid  be  obtained.  For  this  pur- 
23 


266  PRACTICAL    HISTOLOGY. 

pose  it  is  as  usual  first  placed  in  oil  of  cloves,  and 
then  allowed  to  soak  for  half  an  hour  or  more  in 
cacao-butter,  which  is  kept  just  melted  by  a  gentle 
heat.  When  completely  permeated  the  piece  of 
membrane  is  placed  in  the  proper  position  in  a 
cavity  scooped  out  in  a  cake  of  cacao-butter,  and 
after  it  has  firmly  set,  sections  mny  without  diffi- 
culty be  made  of  exquisite  fineness.  They  are  placed 
in  oil  of  cloves — in  winter  it  must  be  very  slightly 
warmed — which  speedily  dissolves  out  the  infiltrated 
cacao-butter,  and  they  may  then  be  mounted  in 
dammar.  But  it  is  better  to  transfer  them  from 
the  oil  of  cloves  to  spirit  again,  and  afterwards  to 
mount  them  in  glycerine,  since  in  this  the  details 
can  be  made  out  with  greater  readiness  than  in  the 
more  highly  refracting  dammar.  For  the  same 
reason  a  saturated  solution  of  acetate  of  potash  is 
sometimes  recommended  as  superior  to  glycerine 
for  mounting  osraic  preparations,  especially  of  the 
retina. 

Preparations  23,  24,  and  25.  Isolation  of  the 
retinal  elements. — Various  processes  are  employed 
for  macerating  portions  of  the  retina  in  order  to 
obtain  its  elements,  either  in  a  completely  isolated 
condition  or  still  partially  connected  with  one 
another.  It  will  be  best,  in  the  first  place,  to  at- 
tempt the  separation  with  a  piece  of  retina  which 
has  been,  in  2  per  cent,  osmic  acid  for  six  or  eight 
hours.  It  must  of  course  be  put  in  perfectly  fresh, 
and  after  the  time  mentioned  it  is  placed  in  water 
for  twenty-four  hours.  It  is  then  allowed  to  mace- 
rate for  a  few  days  in  a  mixture  of  glycerine,  alco- 
hol, and  water  (glycerine  one  part,  alcohol  one  part, 
water  two  parts),  after  which  a  minute  portion  is 
to  be  carefully  broken  up  with  fine  needles  in  a 
drop  of  weak  glycerine,  and,  a  piece  of  hair  having 
been  added,  the  cover-glass  is  superposed,  and  the 
preparation  examined  with  a  dry  objective  and, 
after  fixing  the  cover-glass,  with  an  immersion. 
The  effect  of  the  osmic  acid  is  to  preserve  the  more 


THE    RETINA.  267 

readily  alterable  elements,  such  as  the  rods  and 
cones  and  their  nuclei,  in  a  condition  as  nearly  as 
possible  approaching  that  which  they  possess  during 
life. 

Other  portions  of  fresh  retina  are  to  be  placed,  one 
in  one-eighth  per  cent,  of  bichromate  of  potash  solu- 
tion (for  a  week),  and  the  other  in  ten  per  cent,  of 
chloral  hydrate  solution  (for  two  or  three  days).  The 
portions  so  macerated  are  to  be  teased  out  in  a  drop 
of  their  respective  solutions,  the  usual  expedient 
beinoc  adopted  of  obviating  the  pressure  of  the  cover- 
glass"  by  a  hair.  In  the  one  treated  with  bichromate 
the  Miillerian  fibres,  and  in  fortunate  preparations 
the  ganglion  cells  with  their  processes,  may  be  well 
seen'and  isolated.  The  granules  of  the  inner  nuclear 
layer  are  also  frequently  obtained  with  one  or  both 
processes  extending  from  their  opposite  poles,  but  the 
rods  and  cones  are  for  the  most  part  much  altered 
and  granular  in  appearance,  so  as  hardly  to  be  recog- 
nizable. 

In  the  chloral  hydrate  preparation,  the  last-named 
elements  are  much  better  preserved.  The  external 
segments  of  the  rods,  which  even  in  the  osmic  pre- 
paration tend  for  the  most  part  to  become  altered, 
may  by  this  method  be  frequently  seen  almost 
unchanged,  except  that  the  transverse  striation, 
which  indicates  their  discoid  formation,  is  often 
well  marked.  Most  of  the  other  structures  are  also 
well  preserved,  and  the  Miillerian  fibres  and  ganglion 
cells  are  sometimes  better  seen  than  in  the  bichro- 
mate preparation.  Unfortunately  neither  the  bichro- 
mate nor  the  chloral  specimens  can  be  well  preserved 
without  deterioration. 

Preparations  26  and  27.— The  study  of  the 
retina  cannot  be  considered  complete  until  the  ele- 
ments have  been  examined  in  the  fresh,  unaltered 
condition.  A  small  piece,  taken  from  an  eye  still 
warm  from  the  animal,  should  accordingly  be  broken 
up  as  rapidly  and  finely  as  possible  in  a  drop  of 
serum,  or,  failing  this,  in  a  little  vitreous  humor. 


268  PRACTICAL    HISTOLOGY. 

A  modification  of  this  very  simple  method  consists 
in  allowing  a  small  piece  to  macerate  for  two  or 
three  days  in  weakly  iodized  serum  (see  p.  45)  before 
attempting  the  dissociation,  which  can  then  be  more 
readily  effected. 

Preparation  28. — The  hexagonal  pigment  of  the 
retina  is  seen  in  most  of  the  teased  preparations 
above  described.  In  eyes  that  have  been  hardened 
in  Muller's  fluid  the  layer  often  separates  in  flakes 
of  varying  size,  and  nothing  is  simpler  than  to  re- 
move such  a  piece  with  a  section  lifter,  and  mount 
it  in  glycerine,  so  as  to  exhibit  the  pavemented 
appearance  which  the  cells  present. 

Preparation  29. — On  the  opposite  surface  of  the 
retina  also  a  mosaic-like  appearance  can  be  demon- 
strated by  the  aid  of  nitrate  of  silver,  but  it  is  much 
more  irregular,  and  does  not  depend  upon  the  pre- 
sence of  epithelium  cells,  but  upon  the  flattened-out 
ends  of  the  Miillerian  fibres.  To  show  this  appear- 
ance a  fresh  eye  is  cut  in  half  transversely,  and  the 
vitreous  is  shelled  out  from  the  posterior  half;  this 
is  then  rinsed  in  distilled  water  and  transferred  to 
nitrate  of  silver  solution  (one-half  per  cent.).  After 
a  minute  in  this  it  is  again  rinsed  in  distilled  water, 
and  exposed  in  water  to  the  light.  When  the  sur- 
face is  browned  it  is  removed,  a  piece  of  the  retina 
is  cut  out  under  water,  floated  on  to  a  slide  with  the 
inner,  brown  surface  uppermost,  and  the  water  is 
drained  off  and  replaced  by  glycerine,  when  the  pre- 
paration can  be  covered  and  examined,  care  being 
taken  not  to  let  the  cover-glass  press  upon  the  speci- 
men. 

Preparation  30.  The  Retina  in  the  lower 
Vertebrata. — The  structure  of  the  outer  segments  of 
the  rods,  which  it  is  difficult  to  make  out  in  mammals, 
can  be  seen  easily,  even  with  an  ordinary  high  power,  in 
the  retina  of  amphibia.  With  this  object  the  eye  is  re- 
moved from  a  recently  killed  frog,  cut  across,  and  a  small 
portion  of  the  retina  is  quickly  broken  up  in  vitreous 
humor.  A  piece  of  hair  having  been  added,  the  prepara- 
tion is  covered  and  examined.  Almost  everywhere  the 


THE    LENS.  2G9 

field  of  view  is  strewed  with  large,  clear,  rod-shaped  struc- 
tures, some  straight,  but  many  of  them  bent  and  curved  in 
different  directions,  and  exhibiting  a  distinct  transverse 
striation,  or  even  a  tendency  to  split  up  into  a  number  of 
superimposed  disks,  this  tendency  increasing  as  the  pre- 
paration is  longer  made.  Some  of  them  have  what  looks 
like  a  small  appendage  jointed  on  at  one  end,  but  this  is 
really  the  comparatively  small  inner  segment  of  the  rod. 
The  cones  are  also  very  small  in  comparison,  and  on  that 
account  may  at  first  be"  missed ;  they  are  distinguished  by 
the  possession  at  the  apex  of  the  inner  segment  of  a  small, 
bright,  fatty  globules,  often  of  a  yellow  color.  Most 
likely  a  portion  of  the  hexagonal  pigmented  epithelium 
will  have  come  away  with  the  rest  of  the  retina,  and  in 
consequence  of  the  rupture  of  some  of  the  cells  the  prepa- 
ration will  be  strewed  with  pigment  granules  which,  like 
all  minute  granules  suspended  in  fluid,  exhibit  very  strik- 
ingly the  Brownian  molecular  movement.  Some  of  the 
pigment  cells  may  be  observed  intact,  either  isolated  or 
in  patches.  If  seen  in  profile,  it  may  be  noticed  that  near 
one  surface  (the  outer)  the  cell  is  almost  entirely  free  from 
black  pigment,  while  from  the  other  fine  streamers  of  the 
cell-protoplasm,  dotted  with  pigment  granules,  extend. 
In  their  natural  position  these  pass  between  and  amongst 
the  outer  segments  of  the  rods. 

Preparations  31,  32,  and  33.— The  retina  of  a  bird, 
of  a  reptile  (tortoise),  and  of  a  fish  are  to  be  teased  out 
fresh  in  vitreous,  in  the  same  way  as  that  of  the  frog. 
The  chief  points  of  interest  in  these  preparations  are  the 
ellipsoid  or  lenticular  bodies  in  the  inner  segments  of  the 
rods  (bird  and  amphibian)  and  cones  (bird,  reptile,  and 
amphibian),  the  bright,  fatty  globules  of  different  colors 
in  the  inner  segments  of  the  cones  in  the  tortoise  and 
bird,  and  the  twin  or  double  cones,  especially  large  in  the 
fish's  retina.  The  various  other  points  in  which  the  retina 
in  these  animals  differs  from  that  of  mammals,  may  be 
studied  by  employing  the  same  methods  of  preparations 
as  for  the  mammalian  retina. 


THE  LENS  AND  VITREOUS  HUMOR. 

Preparations  34.     The  lens  fibres.— The  fol- 
lowing will  be  found  the  best  mode  of  isolating  the 

23* 


270  PRACTICAL    HISTOLOGY. 

fibres  of  the  lens,  as  well  as  for  showing  their  ar- 
rangement. Take  the  fresh  eye  of  any  animal — 
that  of  the  ox  or  sheep  for  example — and  cut  it 
across  into  an  anterior  and  a  posterior  half.  Place 
the  anterior  part,  having  removed  what  remains  of 
the  vitreous  humor,  in  J  per  cent,  solution  of  bi- 
chromate of  potash.  Then  scratch  through  the 
posterior  capsule,  which  is  readily  ruptured  and 
curls  away  from  the  lens  proper.  This  can  easily  be 
shelled  out,  and  is  left  in  the  fluid,  the  remainder 
of  the  eye  being  rejected.  The  lens  is  allowed  to 
remain  in  the  bichromate  for  two  or  three  days, 
being  merely  turned  over  once  or  twice.  It  will  be 
found  that  its  substance  tends  both  to  separate  along 
the  radiating  lines  which  mark  the  planes  of  junc- 
tion of  the  ends  of  the  fibres,  and  also  to  peel  into 
concentric  lamellae  like  the  coats  of  an  onion  ;  and, 
if  a  piece  of  one  of  these  lamellae  is  taken  up  with 
the  forceps,  it  will  tear  in  the  direction  of  the  fibres 
from  one  of  the  planes  of  junction  of  the  anterior 
surface  to  the  corresponding  plane  of  the  posterior. 
The  fibres  can  be  readily  separated  with  needles  in 
a  drop  of  the  bichromate  solution.  For  this  pur- 
pose portions  should  be  taken  both  from  the  super- 
ficial and  from  the  more  central  parts  of  the  lens. 
In  many  of  the  superficial  fibres  a  round  or  elon- 
gated nucleus  may  be  detected  at  one  part,  and  since 
the  nuclei  of  adjacent  fibres  are  met  with  in  about 
the  same  region,  when  a  number  of  fibres  are  seen 
together  the  nuclei  lie  in  an  irregular  row.  The 
riband-like  shape  of  the  fibres  may  be  made  out  at 
parts  where  they  are  turned  over  so  as  to  be  seen 
edgeways. 

Preparation  35.  Sections  of  the  Lens, — For 
cutting  sections  of  the  lens  it  is  best  to  harden  it  in 
Muller's  fluid.  The  whole  anterior  half  of  an  eye 
should  be  put  in  this,  the  cornea  having  been  partly 
removed  so  as  to  enable  the  fluid  to  get  freely  to  the 
front  as  well  as  to  the  back  of  the  lens,  but  the 
capsule  is  not  to  be  ruptured.  After  two  or  three 


THE    VITREOUS    HUMOR.  271 

weeks,  vertical  sections  may  be  made,  one  of  which 
should  pass  through  the  centre  from  before  back,  and 
should  be  as  complete  as  possible.  They  are  to  be 
mounted  in  glycerine.  The  lens  must  not  be  put  in 
spirit,  or  at  all  events  not  in  strong  spirit,  to  com- 
plete the  hardening,  for  this  renders  the  tissue, 
especially  the  central  parts,  quite  hard  and  horny, 
and  the  outlines  of  the  fibres  become  obliterated. 

Preparation  36.  The  epithelium  of  the  lens- 
capsule, — This  may  have  been  seen  in  the  antero- 
posterior  section  as  a.  single  row  of  nucleated  cells, 
lying  immediately  behind  the  anterior  part  of  the 
capsule.  To  show  it  on  the  flat  it  is  to  be  stained 
with  nitrate  of  silver.  With  this  object  a  lens  still 
inclosed  in  its  capsule  is  removed  from  a  fresh  eye, 
and,  after  having  been  rinsed  in  distilled  water, 
transferred  for  five  minutes  to  J  per  cent,  nitrate  of 
silver  solution.  It  is  then  again  washed  with  dis- 
tilled water,  and  placed  in  the  light  in  weak  spirit 
(equal  parts  spirit  and  water).  When  brown  it  is 
removed  from  the  light,  and  after  twenty-four  hours 
the  substance  is  hard  enough  to  allow  tangential 
sections  to  be  made  from  the  anterior  surface,  which 
shall  include  the  capsule,  the  epithelium  and  the 
parts  of  the  lens  substance  immediately  subjacent  to 
this.  The  sections  are  mounted  in  glycerine  with  the 
brown  surface  uppermost:  and  through  the  elastic 
capsule,  which  is  not  distinctively  stained,  the  out- 
lines of  the  epithelium-cells  are  clearly  seen.  At 
places  the  silver  solution  may  have  penetrated  to  the 
superficial  lens  fibres,  and  will  be  found  to  have 
stained  the  cementing  substance  between  them. 

Preparation  37.  The  zonule  of  Zinn  and  the. 
hyaloid  membrane  of  the  vitreous  humor.— 
Take  the  anterior  half  of  the  eye  (preserved  in  spirit) 
of  an  albino  rabbit,  and  having  pinned  the  cornea 
downwards  on  a  loaded  cork  under  spirit,  and  re- 
moved the  remains  of  the  vitreous  humor,  gently 
seize  the  lens  with  fine  forceps,  and  draw  it  away 
from  the  iris.  In  doing  this  it  will  drag  with  it  tie 


272  PRACTICAL    HISTOLOGY. 

suspensory  ligament,  the  zonule  of  Zinn,  and  the 
part  of  the  hyaloid  membrane  continuous  with  this, 
so  that  the  separated  lens  appears  girdled  by  a  deli- 
cate, somewhat  crumpled-looking,  membranous  zone, 
closely  adherent  at  its  inner  border  to  the  equator  of 
the  lens,  and  bounded  outwardly  by  a  ragged  mar- 
gin— the  torn  edge  of  the  hyaloid.  Cut  out  with 
fine  scissors  a  segment  of  this  zone,  including  its 
whole  breadth,  and  with  a  section-lifter  transfer  the 
piece  so  removed  to  logwood  solution  (Kleinenberg's). 
When  sufficiently  stained — and  it  stains  very  readily 
— transfer  it  to  a  dish  of  water,  and  from  this  float 
it  on  to  a  slide,  avoiding  all  creases  except  of  course 
the  natural  ones  of  the  zonule.  It  may  then  be 
covered,  and  the  water  in  which  it  is  mounted  re- 
placed by  glycerine.  Or,  instead  of  placing  it  in  the 
water,  it  imiy  be  transferred  from  the  logwood  to 
spirit,  and  then  passed  through  oil  of  cloves  and 
mounted  in  dammar.  These  preparations  exhibit 
well  the  folds  and  striations  of  the  zonule,  and  the 
rounded  corpuscles,  like  white  blood  corpuscles, 
which  are  dotted  here  and  there  over  the  surface  of 
the  hyaloid. 

THE  BLOODVESSELS  OF  THE  EYE. 

For  the  demonstration  of  the  bloodvessels  the  head 
of  an  albino  rabbit  should  be  injected,  a  canula  being 
placed  in  each  carotid,  and  the  two  canulas  con- 
nected to  the  arras  of  a  Y-shaped  tube,  the  stem  of 
which  is  brought  into  communication  by  an  India- 
rubber  tube  with  the  injection  bottle.  After  the 
blood  has  been  driven  out  of  the  vessels  before  the 
flow  of  injection  fluid,  the  neck  of  the  animal,  just 
below  the  place  where  the  canulas  are  inserted,  is 
surrounded  by  a  loop  of  wire,  which  is  drawn  as 
tightly  as  possible  to  prevent  the  escape  of  the  injec- 
tion ;  and  the  pressure  is  then  raised  to  about  four 
inches  of  mercury  and  kept  there  for  some  minutes, 
so  as  to  make  certain  that  all  the  bloodvessels  shall 


BLOODVESSELS    OF    THE    EYE.  273 

be  completely  filled.  The  whole  is  then  allowed  to 
stand  and  become  cold,  that  the  gelatine  may  set, 
after  which  the  eyes  are  to  be  carefully  excised,  and 
placed  in  weak  spirit.  In  twenty-four  hours  this 
may  be  increased  in  strength,  and  in  another  twenty- 
four  or  forty-eight  hours  they  are  to  be  placed  in 
strong  spirit.  When  they  have  been  in  this  a  day 
or  two  the  following  parts  may  be  prepared  : — 

Preparation  387  The  conjunctival  vessels, 
and  the  subeonjunctival  vessels  of  the  sclero- 
tic.— By  making  a  tangential  section  from  the  region 
of  the  corneo-sclerotic  junction,  and  after  passing  the 
piece  so  obtained  through  oil  of  cloves,  mounting  it 
in  dammar  with  the  outer  surface  uppermost,  the 
distribution  of  the  vessels,  both  in  the  conjunctiva 
and,  by  focussing  more  deeply,  those  in  the  sclerotic 
at  and  near  the  margin  of  the  cornea,  is  exhibited. 
Another  plan  consists  in  cutting  away  a  small  piece, 
including  the  whole  thickness  of  both  cornea  and 
sclerotic,  and  mounting  in  a  similar  way.  The 
thickness  and  irregularity  of  the  piece  so  obtained 
is  a  disadvantage,  but,  on  the  other  hand,  the  canal 
of  Schlemm  and  the  other  venous  sinuses  may  be 
observed,  if  the  injection  has  been  a  successful  one, 
by  focussing  still  lower  than  for  the  looped  vessels 
of  the  sclerotic. 

Preparation  39.  Vessels  of  the  choroid  and 
iris. — One  of  the  two  injected  eyes  is  to  be  divided 
by  an  antero-posterior  cut  with  the  razor  into  a  right 
and  a  left  hand.  One  of  the  two  halves,  the  one 
which  does  not  include  the  attachment  of  the  optic 
nerve,  is  first  taken,  and  the  vitreous,  retina,  and 
lens  removed,  so  as  to  clear  the  inner  surface  of  the 
choroid  and  iris.  The  last-named  are  next  to  be 
separated  as  one  piece  of  membrane  from  the  scle- 
rotic. The  piece  so  obtained  is  then  to  be  again 
divided  into  two,  by  another  antero-posterior  cut 
with  the  scissors,  and  the  resulting  halves  are  to  be 
mounted,  after  passing  as  usual  through  oil  of  cloves, 
in  dammar,  the  one  with  the  inner  and  the  other 


274  PRACTICAL    HISTOLOGY. 

with  the  outer  surface  uppermost.  Each  includes, 
of  course,  the  fourth  part  of  the  choroid  coat  with 
some  of  the  ciliary  processes,  and  a  piece  of  the  iris; 
and  with  a  low  power  the  course  and  disposition  of 
the  bloodvessels  in  these  parts  can  be  readily  followed. 
Besides  these  comprehensive  preparations,  separate 
ones  may  be  made  from  the  other  half  of  the  eye  of 
a  portion  of  the  iris  (this  is  rendered  more  instruc- 
tive by  lightly  staining  it  with  logwood),  and  one 
or  two  of  the  ciliary  processes  snipped  off  with  sharp 
scissors,  and  mounted  so  as  to  be  seen  in  profile. 

Preparation  40.  The  vessels  of  the  retina. — 
If  the  other  injected  eye  be  cut  into  an  anterior  and 
a  posterior  half,  and  the  posterior  part  is  examined 
after  removal  of  the  vitreous  humor,  the  bloodvessels 
will  be  seen  spreading  out  from  the  centre  of  the 
colliculus  of  the  optic  nerve.  To  exhibit  their  finer 
distribution  in  the  retina,  a  piece  is  mounted  fiat  in 
dammar  without  previous  staining,  while  to  show 
the  extent  of  their  distribution  in  the  retinal  layers, 
vertical  sections,  which  need  not  be  very  thin,  may 
be  made  from  a  piece  embedded  in  wax-mass  in  the 
ordinary  way,  and  similarly  mounted,  without  stain- 
ing, in  dammar. 


THE    EAR.  275 


CHAPTER    XXI. 

THE  EAR. 

THE  only  parts  of  the  ear  which  require  special 
directions  for  their  preparation  are  the  semicircular 
canals  and  the  cochlea. 

Preparations  1,  2,  and  3.  The  semicircular 
canals. — To  study  the  structure  of  the  membranous 
semicircular  canals,  those  of  the  cartilaginous  fishes, 
e.g.  the  skate,  are  chosen.  •  The  skull,  which  can  he 
readily  cut  with  a  scalpel  or  strong  pair  of  scissors, 
is  opened  quite  anteriorly,  where  it  is  occupied 
merely  by  a  quantity  of  cerebro-spinal  fluid,  and 
the  opening  is  extended  backwards  by  removing 
the  roof  bit  by  bit,  until  the  whole  of  the  upper 
surface  of  the  brain  is  exposed.  Two  thick  carti- 
laginous masses  will  be  seen,  one  on  either  side,  near 
the  posterior  part ;  the  large  auditory  nerves  pass 
through  a  foramen  in  each  into  their  interior. 
These  masses  inclose  the  membranous  labyrinth, 
consisting  in  these  animals  of  utricle,  'saccule,  and 
semicircular  canals,  all  of  large  size,  and  contained 
in  corresponding  cavities  and  canals,  in  the  sub- 
stance of  the  cartilage,  but  of  which  no  trace  can 
at  present  be  made  out.  If,  however,  horizontal 
slices  are  made  with  a  scalpel,  one  of  the  canals  will 
soon  be  exposed,  and  this  can  then  be  followed  in 
both  directions,  cutting  the  cartilage  away  so  as  to 
expose  the  included  membranous  canal  in  its  whole 
length.  It  will  be  found  to  lead  at  either  end  into 
a  large  membranous  bag — the  utricle — with  which 
the  two  other  canals  also  communicate,  and  from 
which  they  can  be  traced  in  the  same  manner. 
Besides  the  utricle,  there  is  another  smaller  mem- 


276  PRACTICAL    HISTOLOGY. 

branous  bag — the  saccule — and  both  contain  a  white, 
pasty,  cretaceous,  otolithic  mass,  which  lies  over  the 
part  to  which  the  nerve  proceeds.  ISTear  one  of  the 
attachments  of  each  semicircular  canal  to  the  utricle 
is  its  dilated  part,  or  ampulla,  and  a  branch  of  the 
auditory  nerve  may  be  seen  proceeding  to  each  of 
these,  and  terminating  abruptly  in  a  forked  thicken- 
ing, which  indents  the  membranous  wall  and  lies 
transversely  to  the  axis  of  the  ampulla. 

The  three  ampullae,  and  the  adjacent  portions  of 
the  semicircular  canals,  are  now  to  be  removed  from 
the  cavities  containing  them,  and  are  to  be  placed, 
one  in  a  weak  solution  of  chromic  acid  (-J-  per  cent.), 
one  in  osmic  acid  (2  per  cent.),  and  the  third  on  a 
slide  in  a  drop  of  endolymph  obtained  from  the 
cavity  of  the  utricle.  The  piece  in  chromic  acid  is 
transferred  to  weak  spirit  after  three  days,  and  in 
twenty -four  hours  more  to  strong  spirit.  After 
another  day  or  two  it  may  be  placed  in  Kleinen- 
berg's  logwood  for  several  hours,  and  then  embedded 
either  in  wax-mass  in  the  ordinary  way,  or  by  the 
cacao-butter  process.  Sections  are  to  be  made  both 
of  the  semicircular  canal  proper,  and  of  the  ampulla, 
opposite  to  and  including  the  entrance  of  the  nerve  ; 
and  the  sections  are  passed  through  oil  of  cloves, 
and  mounted  in  dammar.  The  piece  in  osmic  acid 
is  transferred  after  twenty-four  hours,  first  to  water 
for  two  or  three  hours,  and  then  to  spirit ;  after  a 
day  or  two  in  this  it  may  be  placed  in  oil  of  cloves, 
and  subsequently  permeated  with  and  embedded  in 
cacao-butter.  The  sections,  after  the  cacao-butter 
has  been  removed  by  oil  of  cloves,  and  this  again 
by  spirit,  are  finally  mounted  in  glycerine.  The 
third  piece,  especially  the  part  where  the  nerve 
enters,  is  broken  up  at  once  in  the  drop  of  endo- 
lymph, and  examined  with  a  high  power,  with  the 
view  of  observing  the  two  kinds  of  epithelium  cells — 
columnar  and  spindle-shaped — which  occur  here,  and 
the  stiff,  hair-like  projections  which  are  attached  to 


THE    COCHLEA.  277 

them.     The  demonstration  of  these  structures  pre- 
sents, however,  the  greatest  possible  difficulty. 
Preparation  4.     Sections  of  the  cochlea.— 

On  account  of  the  thinness  of  its  osseous  parietes, 
the  ease  with  which  it  may  be  obtained  separate 
from  the  surrounding  bone,  and  its  comparatively 
large  number  of  spiral  turns,  the  cochlea  of  the 
guinea-pig  offers  far  greater  facilities  for  study,  and 
especially  for  the  preparation  of  sections,  than  that 
of  any  other  animal.  The  following  is  the  mode  of 
finding  and  procuring  it:  In  the  recently-killed 
animal  the  aperture  of  the  mouth  is  prolonged  back- 
wards on  either  side,  by  cutting  through  the  cheeks 
and  temporal  muscles  with  strong  scissors.  The 
lower  jaw  is  then  seized  and  forcibly  torn  away 
from  the  rest  of  the  head,  so  that  the  base  of  the 
skull  is  exposed.  Here  will  be  seen  on  either  side, 
just  behind  the  fossa  for  the  articulation  of  the  con- 
dyle  of  the  jaw,  a  large  white  bony  projection — the 
tympanic  bulla.  This  is  not  yet  to  be  opened,  but 
the  cartilaginous  external  auditory  meatus  is  first  cut 
tli rough,  and  with  the  aid  of  bone-forceps  or  strong 
scissors,  the  bulla  in  question,  together  with  the 
petrous  bone  to  which  it  is  attached,  separated  from 
the  rest  of  the  skull.  In  a  young  animal  this  can 
be  readily  effected,  simply  by  inserting  a  strong 
blunt  instrument  into  the  base  of  the  skull  just  in 
front  of  the  bulla,  and  using  it  as  a  lever,  raising 
the  bone  and  forcing  it  away  from  its  attachments. 
The  bones  of  either  side  being  thus  removed,  the  ad- 
hering soft  parts  are  cleared  away,  and  the  bulla  is 
broken  open  at  its  most  prominent  part.  On  now 
looking  into  the  cavity  there  will  be  noticed,  on  one 
side  the  delicate  tympanic  membrane  stretching  over 
the  end  of  the  external  meatus,  with  the  handle  of 
the  malleus  attached  to  it,  and  on  the  opposite  wall 
a  well-marked  conical  projection,  indeed,  its  bony 
wall  is  so  thin  that  it  is  possible  to  count  the  number 
of  turns  (four)  which  it  presents.  By  cutting  the 
bulla  round  with  strong  scissors,  the  two  parts — one 
24 


278  PRACTICAL    HISTOLOGY. 

including  the  tympanic  membrane,  and  the  other 
the  cochlea — are  separated  from  one  another,  and  the 
membrane  part  may  at  once  be  dropped  into  weak 
spirit  and  put  aside  to  be  subsequently  stained  and 
mounted.  From  the  other  part  as  much  as  possible 
of  the  substance  of  the  petrous  bone  is  snipped  away, 
bit  by  bit,  from  around  the  base  of  the  cochlea  with 
scissors  or  bone  forceps,  but  great  care  should  be 
taken  in  approaching  the  cochlea  itself,  as  this  is 
very  readily  split.  When  the  surrounding  bone  has 
been  in  this  way  removed,  the  cochleas  are  dropped 
into  Miiller's  fluid.  In  this  they  are  to  be  left  for  a 
week  or  fortnight — even  a  longer  immersion  will  do 
them  no  harm — until  the  sort  structures  in  the  in- 
terior are  somewhat  hardened.  The  process  is  then 
completed,  whilst  the  bone  is  at  the  same  time 
softened  by  transferring  the  cochleae  to  a  saturated 
solution  of  picric  acid.  When  the  bone  is  com- 
pletely softened,  a  process  which  is  much  facilitated 
by  frequent  disturbance  of  the  fluid,  the  cochleas  are 
transferred  to  weak  spirit  (half  water),  and  in 
twenty-four  hours  more  to  strong  spirit.  After 
beini>;  in  this  for  two  or  three  days  they  are  ready  for 
embedding.  The  best  mass  to  use  for  this  purpose 
is  a  mixture  of  wax  and  cacao  butter,  equal  parts  of 
each.  One  of  the  prepared  cochleas  is  fixed  by  a  pin 
in  the  embedding  box  in  such  a  position  that  the 
plane  in  which  the  sections  are  made  shall  be  exactly 
parallel  with  the  axis  of  the  cone  which  the  cochlea 
forms,  and  the  cacao-butter  and  wax,  previously 
melted  and  thoroughly  mixed,  are  poured  into  the 
mould.  When  the  mass  has  become  hard,  sections 
may  be  made ;  the  first  ones  will  include  only  the 
large  basal  turn  of  the  cochlea,  then  the  higher  turns 
will  all  be  included  in  succession,  until  at  last  the 
modiolus  is  reached.  All  the  sections  which  have 
been  made  up  to  this  point  may  be  rejected.  Great 
care  must  now  be  taken  to  make  thin  and  complete 
sections  of  this  central  part.  They  will  of  course  be 
triangular  in  shape,  with  a  rounded  apex;  the  sec- 


THE    COCHLEA.  279 

tion  of  the  modiolus  occupies  the  axis  from  the  base 
to  near  the  apex  of  the  triangle,  and  on  either  side 
are  the  sections  of  the  successive  spiral  turns.  Not 
more  than  three  or  four  complete  axial  sections  can 
l>e  obtained  from  each  cochlea.  The  razor  and  the 
surface  of  the  cake  should  be  wetted  with  oil  of 
cloves  instead  of  spirit.  It  may  be  applied  by  means 
of  a  large  camel-hair  brush,  and  its  use  is  that  it 
renders  the  parts  more  coherent,  and  tends  to  prevent 
the  organ  of  Corti  from  breaking  away  from  the 
basilar  membrane,  which  it  is  otherwise  apt  to  do. 
The  sections  are  examined,  unstained,  with  a  low 
power,  and  if  the  organ  of  Corti  is  sufficiently  com- 
plete they  are  at  once  transferred  to  an  alcoholic 
staining  fluid.  One  of  the  best  is  a  weak  solution  of 
acetate  of  rosaniline,  but  if  this  is  not  at  hand,  some 
other  aniline  dye,  such,  for  example,  as  eosin  (see 
Appendix),  may  be  employed.  When  stained  the 
sections  are  again  placed  in  oil  of  cloves,  and  finally 
mounted  in  dammar. 

Preparations  5  and  6.  Teased  preparations 
of  the  cochlea. — Successful  sections  will  show 
the  general  position  and  relations  of  the  rods  and 
other  parts,  and  to  a  certain  extent  the  individual 
elements.  But  only  a  profile  view  can  in  this  way- 
be  obtained,  and  since  the  minute  structure  of  the 
elements  composing  the  organs  of  Corti  can  only 
be  properly  seen  when  isolated,  it  is  necessary  to 
prepare  other  cochleas  with  this  object  in  view. 
Another  animal  is  accordingly  sacrificed,  and  the 
cochleas  removed  as  before.  One  placed  in  a  2  per 
cent,  solution  of  osraic  acid  ;  the  other  in  a  J  per 
cent,  solution  of  bichromate  of  potash  ;  but,  before 
dropping  them  into  their  respective  fluids,  the  bony 
wall  must  be  scraped  through  here  and  there  with 
a  scalpel,  so  that  the  fluid  "shall  at  once  penetrate 
to  the  interior  of  the  turns.  After  two  days  the 
cochleas  may  be  further  prepared  in  the  following 
way : — 

The  uppermost  turn  is  broken  or  snipped  off  with 


280  PRACTICAL    HISTOLOGY. 

scissors,  placed  in  a  drop  of  water  on  a  slide,  the 
shell  of  bone  which  forms  the  cupola  and  outer  wall 
removed,  and  the  piece  of  lamina  spiralis  examined 
with  a  low  power  (without  covering  the  preparation) 
in  order  to  learn  to  recognize  the  structures  which 
lie  on  it.  The  glass  slide  is  then  removed  to  the 
dissecting  microscope,  and  with  very  iine  needles 
the  lamina  spiralis  is  separated  from  the  columella, 
which  is  then  rejected.  Next  all  the  parts  on  the 
lamina,  but  especially  the  row  of  rods  of  Corti,  to 
which  the  hair-cells  as  a  rule  cling,  are  broken  up 
finely, -but  at  the  same  time  slowly  and  carefully, 
the  preparation  being  examined  now  and  again  with 
the  highest  power  which  it  is  safe  to  use  without  a 
cover-glass.  One  of  the  chief  difficulties  is  apt  to 
arise  from  portions  of  the  tissue  sticking  to  the 
needles  ;  if  this  is  the  case,  pieces  of  glass  rod  drawn 
out  to  a  fine  point  may  be  substituted.  When  the 
more  important  parts  have  been  broken  up  pretty 
completely,  any  thick  pieces  of  tissue  unimportant 
to  the  present  observation,  such  as  bits  of  bone,  or 
periosteum,  bundles  of  rnedullated  nerve  fibres,  &c., 
should  be  picked  out,  and  then  a  cover-glass  laid  on 
and  the  preparation  examined.  To  preserve  either 
preparation  permanently  glycerine  may  be  allowed 
to  diffuse  in  at  the  edge  of  the  cover-glass ;'  but  the 
bichromate  specimen  should  first  be  treated  with  a 
drop  of  carmine  solution,  so  that  the  elements  are 
somewhat  stained,  otherwise  they  will  be  rendered 
too  transparent  by  the  glycerine. 

In  this  way  a  number  of  specimens  may  be  obtained 
from  each  cochlea — proceeding  from  above,  down, 
and  preparing  turn  after  turn  ;  and  careful  sketches 
should  be  made  of  the  different  structures  met  with, 
and  their  arrangement  with  regard  to  one  another. 
It  will  be  found  that  the  osmic  preparations  serve 
best  for  showing  the  lamina  reticularis  and  the 
lamina  basilaris,  and  the  bichromate  preparations 
for  the  hair-cells  and  the  membrana  tectoria ;  the 
other  structures  are  almost  equally  well  seen  in  both 


THE    OLFACTORY    ORGAN.  281 

kinds  of  preparations.  The  large  fat  droplets  in 
some  of  the  epithelium  cells  of  the  uppermost  turn 
are  peculiar  to  the  guinea-pig,  as  is  also  the  arched 
projection — seen  in  the  sections — at  the  part  where 
these  cells  are  found.  The  fat  drops  are  stained 
black  in  the  osmic  preparation. 

THE  OLFACTORY  ORGAN. 

Preparations  1-3.— Small  pieces  of  the  upper 
turbinate  bones,  or  from  the  upper  (olfactory)  region 
of  the  septum  nasi,from  the  dog  or  rabbit,  are  placed, 
one  piece  in  one-sixth  per  cent  of  chromic  acid  solu- 
tion, a  second  in  one-fourth  per  cent,  of  bichromate 
of  potash,  and  a  third  in  one  per  cent,  of  osmic  acid. 
The  one  in  chromic  acid  may  remain  a  week,  when 
it  is  transferred  to  weak  spirit  and  in  twenty-four 
hours  more  to  strong  spirit.  After  a  day  or  two  in 
this,  vertical  sections  are  prepared  from  it. 

The  other  two  pieces  are  examined  after  forty-eight 
hours'  maceration,  small  pieces  of  the  mucous  mem- 
brane being  teased  out  so  as  to  isolate  the  epithelium 
cells  (both  columnar  and  spindle-shaped),  and  if  pos- 
sible, especially  in  the  osmic  preparations,  to  study 
the  connection  of  their  branching  lower  ends  with 
subjacent  structures.  These  preparations  can  be  pre- 
served with  glycerine,  the  bichromate  one  being 
stained  with  logwood. 

Preparations  4  and  5. —Teased  preparations 
should  also  be  made  of  the  olfactory  mucous  mem- 
brane of  the  frog  or  newt.  Having  cut  oft'  the  head 
of  the  animal,  and  slit  up  the  nostrils  with  fine  scis- 
sors, place  it  in  a  quantity  of  one-fourth  per  cent, 
solution  of  bichromate  of  potash.  After  two  days' 
preparations  of  the  epithelium  from  both  the  anterior 
and  posterior  part  of  the  passage  may  be  made.  The 
cells  are  obtained  with  the  greatest  ease,  by  scraping 
the  mucous  surface  with  the  point  of  a  scalpel,  and 
shaking  out  the  material  in  a  drop  of  water  on  a 
slide.  A  piece  of  hair  is  added,  and  the  preparation 

24* 


282  PRACTICAL    HISTOLOGY. 


covered  and  examined.  In  the  portion  obtained  from 
near  the  anterior  nares  ordinary,  columnar,  ciliated 
epithelium  cells  will  be  seen.  In  that  form  the  true 
olfactory  part  the  cells,  although  many  of  them  are 
destitute  of  cilia,  and  in  addition  to  the  columnar, 
elements  spindle-shaped  (olfactory)  cells  are  met  with 
which  are  provided  with  a  bunch  of  stiff-looking, 
hair-like  processes,  resembling  the  similar  appen- 
dages of  the  auditory  epithelium. 

THE  GUSTATORY  ORGANS. 

For  studying  the  taste-buds,  the  foliated  papillae 
which  are  found  on  either  side  of  the  base  of  the 
rabbit's  tongue  are  used.  To  obtain  them  the  tongue 
is  cut  out  entire  from  the  recently-killed  animal, 
when  the  little  .oval  patches  marked  with  transverse 
ridges  may  readily  be  found  (Fig.  32,  p,  p).  They 

Fig.  32. 


\ 
P 

Tongue  of  rabbit,  seen  from  above. 
p,  p,  Papillae  folialse. 

are  snipped  off  with  curved  scissors,  and  one  is 
dropped  into  a  mixture  of  equal  parts  of  spirit  and 
one-half  per  cent,  of  chromic  acid,  and  the  other  is 
placed  in  one  per  cent,  of  osmic  acid.  After  two 
days  the  spirit  and  chromic  mixture  is  to  be  ex- 
changed for  strong  spirit,  and  in  twenty-four  hours 
more,  the  piece  of  tissue  may  be  embedded  so  as  to 
cut  sections,  which  should  be  as  thin  as  possible, 
vertical  to  the  surface  of  the  mucous  membrane, 


TASTE-BUDS.  283 

and  across  the  direction  of  the  ridges.     The  sections 
are  stained  and  mounted  as  usual. 

The  piece  that  was  placed  in  osmic  acid  may  be 
used  at  the  end  of  forty-eight  hours.  In  the  first 
place  two  or  three  sections  are  to  be  obtained  like 
those  made  from  the  other  piece,  without  however 
embedding  the  tissue,  but  simply  holding  it  in  the 
fingers  or  in  a  piece  of  split  cork.  One  such  section 
is  to  be  placed  in  a  drop  of  water  on  a  slide,  and  an 
attempt  made  with  needles,  under  the  dissecting 
microscope,  to  separate  some  of  the  taste-buds  from 
the  surrounding  epithelium.  For  this  purpose  the 
needles  must  be  very  fine,  sharp,  and  clean,  and  the 
lens  used  as  high  as  is  consistent  with  convenience 
of  manipulation.  When  one  or  more  taste-buds  have 
been  thus  separated,  the  rest  of  the  section  is  re- 
moved, and  the  isolated  buds  are  broken  up  as  com- 
pletely as  possible  into  their  constituent  cells.  The 
specimen  may  then  be  covered,  and  a  drop  of  glyce- 
rine allowed  to  diffuse  in  under  the  edge  of  the 
cover;  after  which  an  examination  of  the  [(repara- 
tion may  be  made,  at  first  with  the  ordinary  high 
power,  and  afterwards  with  an  immersion  objective. 


APPENDIX. 


Method  of  measuring  an  object  under  the 
microscope. — If  while  the  one  63^6  looks  down  the  tube 
the  other  is  allowed  to  remain  open,  an  image  of  the 
object  will  appear  projected  on  the  table  at  the  side  of 
the  microscope,  and  it  is  not  difficult  to  mark  off,  upon  a 
sheet  of  paper  placed  here,  the  points  between  which  the 
measurements  are  to  be  taken.  The  preparation  is  then 
removed,  and  a  stage  micrometer  is  substituted  for  it, 
the  parts  of  the  microscope  being  left  in  the  same  condi- 
tion as  before.  The  stage  micrometer  is  a  glass  slide  on 
which  fine  equidistant  parallel  lines  have  been  ruled  with 
a  diamond.  The  distance  between  the  lines  is  marked  on 
the.  slide  ;  it  is  generally  either  the  T<jotn  an(*  Wo otn  Parfc 
of  an  inch,  or  the  y^th  and  T^oth  part  of  a  millimetre. 
The  lines  are  observed  with  the  microscope  in  the  same 
way  as  the  object,  and  their  image  can  of  course  be  simi- 
larly projected  upon  a  sheet  of  paper  and  there  marked 
down.  The  distances  between  the  lines  being  known,  it 
is  easy,  by  comparison  of  the  two  markings,  to  find  out 
the  distance  between  the  opposite  points  of  the  object. 

Some  microscopes  are  provided  with  an  eye-piece 
micrometer  (Fig.  33).  This  is  an  ordinary  ocular  witli 
a  flat  piece  of  glass  (m)  having  a  scale  ruled  upon  it  by 
a  diamond,  inserted  between  the  field-glass  and  eye-glass. 
The  value  of  the  divisions  of  the  scale  should  lie  deter- 
mined once  for  all  for  each  objective  by  observation  of  a 
stage  micrometer  (see  Fig.  34),  the  tube  of  the  micro- 
scope being  fully  drawn  out,  and  should  be  marked  on 
the  ocular;  and  in  subsequently  using  it  for  measurement 
all  that  is  necessary  is  to  see  how  many  divisions  of  the 
scale  the  object  under  examination  covers.  Thus,  sup- 
posing it  had  been  found  by  examination  of  a  stage 
micrometer  that  with  the  high  power  objective  and  the 


286  APPENDIX. 

tube  drawn  out  each  division  of  the  eye-piece  micro- 
meter was  worth  TsVu  inch,  any  object  which  when  viewed 
by  the  same  objective  and  length  of  tube  took  up  three 
divisions  of  the  eye-piece  micrometer  would  measure 
i5Svffths  or  gjtfth  of  an  inch. 

The  advantage  of  the  ej^e-piece  micrometer  is  that 
when  its  values  are  once  ascertained  the  size  of  an  object 
can  be  read  off  at  once. 

Fig.  33. 


Ocular  micrometer,  natural  size. 

Part  of  the  side  is  represented  as  broken  away  to  show  the  field-glass  at  the 
bottom,  and  the  micrometer-glass,  rn,  a  little  below  the  middle  The  collar, 
r  serveH  to  vary  the  distance  of  the  eye-glass  from  the  microniPter. 

Determination  of  the  magnifying  power  of  a 
microscope.  —  The  magnifying  power  of  a  microscope 
is  determined  by  comparing  the  distance  between  the 
lines  of  the  stage  micrometer,  as  they  appear  imaged 
upon  the  paper,  when  this  is  exactly  ten  inches1  from  the 
eye,  with  the  known  interval  between  them.  For  instance, 
if,  with  the  high  power  objective  and  the  ordinary  ocular, 
the  interval  of  ^1  of  an  inch  of  the  micrometer  was 


1   The  ordinary  distance  of  distinct  vision. 


APPENDIX. 


287 


represented  on  the  paper  by  a  space  of  half  an  inch,  this 
interval  is  magnified  as  many  times  as  the  j^V^th  of  an 
inch  will  go  into  half  an  inch,  that  is  to  say,  500  times ; 
and  every  other  object  under  similar  conditions  is  magni- 
fied to  a  like  extent. 

Fig.  34. 


Lines  of  stage  micrometer  viewed  with  an  ocular-micrometer. 
The  finer  lines  are  those  of  the  stage  micrometer  ;  about  eighteen  of  the  lines  of 
the  ocular  micrometer  are  comprised  in  oue  of  the  larger  intervals  between 
them,  so  that   if  these  intervals  represent  _^_  inch,  the  subdivisions  of  the 
ocular  micrometer  will  represent      l_._  inch. 

The  enlargement  thus  obtained  may  be  determined 
once  for  all  for  each  objective,  the  same  ocular  being 
used  and  the  tube  being  drawn  out  to  the  same  extent — 
say,  to  the  full  length — in  each  case,  and  scales  may  be 
made  representing  the  intervals  between  the  micrometer 
lines  under  the  different  powers.  For  purposes  of  measure- 
ment it  will  then  only  l)e  necessar}7  to  compare  the  pro- 


288 


APPENDIX. 


jected  image  of  an  object  with  the  scale  which  was  made 
under  like  conditions,  without  again  making  use  of  the 
stage  micrometer. 

Mode  of  drawing  microscopic  objects. — The 
most  convenient  and  ready  way  of  delineating  an  object 
is  to  sketch  it  with  the  free  hand  on  paper  placed  at  the 
right  of  the  microscope,  the  left  eye  being  applied  to  the 
tube.  But  if  perfect  accuracy  of  size  and  relations  of 
parts  are  desired,  the  outlines  are  first  traced  with  a 
camera  lucida.  The  simplest  for  ordinary  use  is  that  of 
Zeiss  (Fig.  35),  which  is  a  combination  of  two  prisms  so 

Fig.  35. 


Camera  lucida,  for  tracing  the  outlines  of  an  object  without  tilting  or 
otherwise  disturbing  the  microscope. 

The  metal  ring  fits  on  to  the  upper  end  of  the  microscope  tube,  and  the  aperture, 
a,  is  placed  immediately  over  the  eye-glass,  this  part  of  the  camera  being 
somewhat  more  depressed  than  is  represented  in  the  figure. 

arranged  that  when  placed  above  the  ocular  the  surface 
of  the  table  in  front  of  the  microscope  is  seen  at  the  same 
time  as  the  object,  the  image  of  the  one  being  superposed 
on  that  of  the  other.  The  paper  for  drawing  is  placed  on 
this  part  of  the  table,  supported  upon  an  inclined  plane 
of  wood,  and  the  main  outlines  are  traced  out  on  it,  the 
details  and  shading  being  afterwards  filled  up  without 
the  camera. 

The  lines  of  the  stage  micrometer  can  very  easily  be 
traced  in  the  same  way  by  aid  of  the  camera  lucida. 


APPENDIX. 


289 


Mode  of  counting  the  blood  corpuscles. — In 

order  to  separate  the  corpuscles  and  prevent  coagulation, 
the  blood  used  is  first  diluted  to  a  definite  extent— say  a 
hundred  times — with  a  10  per  cent,  solution  of  sulphate 
of  soda.  The  mixing  can  be  performed  in  a  measuring 
glass  if  the  blood  is  in  sufficient  quantity,  but  if  only  a 
small  drop  is  obtainable,  snch  for  example,  as  is  got  by 
pricking  the  finger,  the  mixer  shown  in  Fig.  36,  B,  may 
be  used.  This  consists  of  a  capillary  tube  terminating 

Fig.  36. 


Malassez's  apparatus  for  counting  the  blood  corpuscles. 

A,  Capillary  tube  for  receiving  the  diluted  blood  ;  B,  pipette  for  m'.xing  a 
small  quantity  of  bio  >d  with  the  diluting  fluid 

in  a  bulb,  the  capacity  of  the  bulb  between  the  marks  I 
and  101  being  exactty  100  times  that  of  the  tube  from  its 
point  to  the  mark  1.  A  small  glass  ball  is  inclosed  in 
the  bulb,  and  serves  by  its  movements  to  facilitate  the 
mixing.  The  capillary  tube  is  allowed  to  fill  with  blood 
as  far  as  the  mark  1 ;  sulphate  of  soda  solution  is  then 
sucked  up  as  far  as  the  mark  101.  As  it  passes  in,  it  of 
course  pushes  the  blood  before  it  into  the  bulb,  and  the 
two  are  there  thoroughly  mixed  by  gentle  agitation. 
The  next  thing  is  to  count  the  corpuscles  in  a  known 
25 


290  APPENDIX. 

quantity  of  the  mixture.  The  most  convenient  plan  is 
that  of  Hay  em  and  Nachet.  A  slide  is  used  having  a 
glass  ring,  1  millimetre  in  depth,  cemented  on  to  its  upper 
surface.  A  drop  of  the  mixture,  not  enough  to  fill  the 
cell  so  formed,  is  placed  in  the  middle  of  the  ring,  and  a 
perfectly  flat  cover-glass  is  so  laid  on  that  the  drop  touches 
and  adheres  to  it  without  reaching  the  sides  of  the  cell. 
The  slide  is  placed  on  the  microscope,  and  as  soon  as  the 
corpuscles  have  settled  down  to  the  bottom  of  the  drop 
the  number  in  a  definite  area  is  counted.  If  the  area 
chosen  is  J  of  a  millimetre  square,  this  will  give  the  num- 
ber which  were  contained  in  £  millimetre  cube  of  the 
mixture,  and  multiplying  this  by  the  number  of  times  the 
blood  was  diluted,  the  result  will  be  the  number  of  cor- 
puscles in  J  millimetre  cube  of  blood. 

Areas  of  J  millimetre  square  might  be  marked  with  a 
diamond  (like  the  lines  of  a  stage  micrometer)  upon  the 
centre  of  the  slide,  and  subdivided  into  smaller  squares 
to  facilitate  the  counting.  But  it  is  found  more  conve- 
nient to  measure  them  off  by  using  an  ocular  micrometer 
similar  to  the  one  shown  in  Fig.  33,  but  with  a  large 
square  subdivided  into  smaller  squares  substituted  for 
the  scale  on  the  micrometer  glass,  m.  To  obtain  the  de- 
sired value  for  the  large  square,  a  stage  micrometer,  ruled 
in  parts  of  a  millimetre,  is  placed  under  the  microscope, 
and  by  adjusting  the  draw-tube,  the  sides  of  the  square 
on  the  ocular  are  made  exactly  to  subtend  the  interval  of 
\  millimetre  on  the  stage  micrometer.  A  mark  is  then 
made  on  the  tube  to  indicate  the  extent  to  which  it  is 
drawn  out,  and  whenever  an  examination  is  made  the 
draw-tube  is  always  adjusted  to  this  mark,  the  same  ob- 
jective (one  of  moderate  power)  being  of  course  used  in 
every  case. 

By  another  method — that  of  Malassez — a  little  of  the 
mixture  of  blood  and  sulphate  of  soda  is  transferred  to  a 
very  fine  flattened  capillary  tube  (Fig.  36  A)  the  capacity 
of  a  given  length  of  which  is  ascertained  previously  and 
marked  on  the  slide  to  which  the  tube  is  fixed.  Thus  in 
the  capillary  tube  shown  in  the  figure,  a  length  of  400 
micro  millimetres1  represents  the  TFV.s  part  of  a  cubic 

1  A  micro-millimetre  (/*)  is  the  one-thousandth  part  of  a  milli- 
metre. 


APPENDIX.  291 

millimetre  of  the  mixture.  The  counting  is  performed 
with  the  aid  of  a  squared  ocular  micrometer,  the  micro- 
scope as  before  having  been  so  arranged  by  observation 
of  a  stage  micrometer  that  the  side  of  the  square  shnll 
have  the  value  of  one  of  the  lengths  (400  ,«,  for  example) 
marked  on  the  slide.  The  result  of  the  counting  gives 
the  number  of  corpuscles  in  a  known  quantity  (y^.-g-  cub. 
mill.)  of  the  mixture,  and  the  number  in  the  same  volume 
of  blood  can  readily  be  deduced. 

Microtomes. — When  it  is  desired  to  obtain  a  number 
of  perfectly  even,  consecutive  sections  of  an  organ,  of  the 
spinal  cord  for  instance,  a  section-instrument  or  micro- 
tome may  be  employed.  One  of  the  first  of  these  to  be 
introduced  was  that  devised  by  Mr.  Stirling  (Fig.  3t), 


Stirling's  microtome. 

and  most  of  the  other  instruments  are  modelled  on  the 
same  principle.  It  consists  of  a  hollow  brass  cylinder 
with  a  broad  metal  plate  (p)  fitted  over  the  top,  and  a 
long,  finely  cut  screw  (.s-)  working  in  the  lower  end,  and 
serving  to  push  a  brass  plug  upwards  in  the  tube.  The 
instrument  is  clamped  at  the  edge  of  the  table  by  the 
screw  cs. 


292  APPENDIX. 

The  tissue  to  be  cut  is  embedded  in  the  brass  cylin- 
der in  much  the  same  way  as  in  an  ordinary  embedding 
trough.  A  little  of  the  embedding  mass  is  first  poured 
in  ;  when  this  has  begun  to  set  the  tissue  is  placed  upon 
it,  and  the  cylinder  is  then  filled  up  with  more  embedding 
mass  so  that  the  object  is  completely  inclosed.  It  is 
better  to  use  a  mixture  of  paraffin  and  hog's  lard  (5  parts 
paraffin  to  1  part  lard)  as  an  embedding  mass,  since  the 
ordinary  wax  and  oil  mixture  tends  in  cooling  to  shrink 
away  from  the  sides  of  the  tube. 

In  cutting  the  sections  the  metal  plate  p  serves  to 
direct  the  razor,  so  that  it  moves  in  a  perfectly  even  plane. 
By  turning  the  screw  .s  the  plug  of  embedding  mass  with 
the  included  tissue  is  caused  to  project  very  slightly 
above  the  plane  of  the  plate,  so  that  when  the  razor,  wetted 
as  usual  with  spirit,  is  carried  over  this,  a  section  is  ob- 
tained varying  in  thickness  according  to  the  extent  to 
which  the  screw  was  turned.  In  like  manner  a  large 
number  of  consecutive  slices  may  be  made.  But  it  is 
doubtful  whether,  with  this  or  any  other  microtome,  it  is 
possible  to  get  sections  as  thin  as  they  can  be  made  with 
the  free  hand. 


Simple  form  of  microtome  for  holding  in  the  hand  whilst  cutting  sections. 

iMjnire  88  shows  a  smnller  instrument  (Ranvier's), 
differing  from  the  other  one  chiefly  in  the  fact  that  it  is 
held  in  the  hand  whilst  the  sections  are  cut.  in  place  of 


APPENDIX.  293 

being  damped  to  the  table.  Its  smaller  size  renders  it 
more  handy  to  work  with,  and  if  it  is  necessary  to  inter- 
mit the  slicing  for  any  length  of  time,  the  embedded  tis- 
sue can  be  preserved  without  injury  by  inverting  the  in- 
strument into  a  beaker  of  spirit. 

Dr.  Rutherford's  freezing  microtome  (F"ig.  39)  is 
designed  to  enable  sections  to  be  made  of  a  tissue 
hardened  by  freezing.  The  instrument  can  also  be  used 
as  an  ordinary  microtome.  It  may  be  described  as  an 

Fig.  39. 


o 
Rutherford's  freezing  microtome. 

B,  Plate  of  gun- metal,  with  aperture,  A.  leading  into  well  of  micro  tone;  0,  lower 
t'nd  of  cylinder  in  which  the  screw,  D,  works,  moving  a  brass  plug  up  or  down  ; 
E,  indicator  for  showing  the  fraction  of  a  revolution  imparted  to  the  screw;  0, 
trough  to  hold  the  freezing  mixture,  with  H,  tube  to  c  induct  away  the  water 
produced  by  the  melting  of  ihe  ice ;  F,  clamp  to  fix  the  instrument  to  a  table. 

improved  form  of  Stirling's  microtome  with  the  addition 
of  a  capacious  metal  trough  which  surrounds  the  upper 
part  of  the  cylinder.  The  trough  is  filled  with  a  freezing 
mixture  (snow  or  pounded  ice  and  salt,  equal  parts). 
Into  the  well  or  tube  of  the  microtome  is  poured  a  thick 
solution  of  gum,  which  soon  begins  to  freeze  at  the  peri- 
phery. The  tissue  to  be  cut  (which  may  be  either  fresh, 
or  hardened  by  one  of  the  ordinary  methods,  and  should 
preferably  be  soaked  in  gum  for  some  hours  previously), 
is  placed  in  the  gum  and  held  there  until  fixed  by  the 
advancing  congelation,  and  when  the  whole  is  uniformly 


294  APPENDIX. 

hard,  sections  are  cut  as  with  the  ordinary  instrument 
except  that  the  razor  Used  must  be  dry,  not  wetted  with 
spirit.  The  sections  are  placed  in  weak  spirit  (1  part 
spirit  2  parts  water)  if  a  hardened  tissue,  in  salt  solution 
if  fresh,  to  dissolve  out  the  gum,  and  they  can  then  be 
mounted  in  any  desired  manner.1 

Gum  is  useful  for  the  freezing  process  because  it  ac- 
quires a  cheesy  consistence  when  frozen,  in  place  of  be- 
coming hard  and  crystalline  (Urban  Prit chard), 

Microphotographic  Apparatus. — Photography  is 
every  day  coming  more  into  use  for  obtaining  images  of 
microscopic  objects.  The  mode  of  application  consists 
in  the  adjustment  of  a  photographic  camera  to  the  tube 
of  the  microscope  (which  it  is  better  to  divest  of  its  eye- 
piece); the  image  formed  by  the  objective  is  received  and 
focussed  upon  the  ground-glass  plate  of  the  camera,  the 
objective  of  the  microscope  representing  in  fact  the  lens 
of  the  ordinary  camera.  Either  wet  or  dry  sensitized 
plates  are  employed,  and  the  mode  of  exposing  the  plate 
and  all  the  subsequent  processes  of  developing,  fixing, 
and  printing  are  in  every  respect  the  same  as  in  ordinary 
photography.  Jt  will  be  sufficient,  therefore,  in  this  place 
to  describe  a  convenient  form  of  apparatus  (Fig.  40),  and 
the  manner  of  obtaining  an  image  of  the  microscopic  ob- 
ject;  an  account  of  the  subsequent  procedure,  and  the 
precautions  it  is  necessary  to  take,  will  be  found  in  recog- 
nized standard  works  on  photography. 

Since  it  may  often  be  required  to  photograph  wet  pre- 
parations, the  microscope  should  be  kept  vertical,  and  the 
camera  must  therefor  be  supported  above  the  microscope. 
This  is  done  by  means  of  a  strong  mahogany  frame  con- 
sisting of  a  stand,  .?£,  upon  which  the  microscope  is  placed 
and  four  vertical  pillars  rigidly  connected  below  to  the 
stand,  and  above  to  one  another  by  cross-pieces.  Two 
brass  rods,  r,  one  on  either  side,  run  vertically  between 
the  stand  below  and  the  cross-piece,  and  serve  more  imme- 
diately to  support  the  camera,  r,  which  is  capable  of  slid- 
ing up  and  down  on  the  rods  and  can  be  fixed  by  screw- 
clamps  at  any  height. 

The  camera  should  be  lightly  constructed,  with  a  bel- 

1  For  a  more  complete  description  sec  Rutherford, 
Practical  Histology."  2d  edition,  L87G. 


295 


Microphotographic  apparatus.     One-seventh  the  natural  size. 

lows-adjustment  gradually  tapering,  and  fitted  below  with 
a  cloth-lined  tube  which  exactly  fits  over  the  microscope 


296  APPENDIX. 

tube,  to  which  it  is  clamped  by  the  screw  d.  Just  above 
the  tube  is  placed  the  "stop"  for  shutting  the  light  off' at 
any  moment  from  the  camera  plate;  it  consists  of  a  light 
hinged  flap,  which,  by  turning  a  small  handle  (not  shown 
in  the  figure),  may  be  made  to  fall  over  the  aperture. 

The  microscope  is  put  in  the  centre  of  the  stand,  being 
held  firmly  in  place  by  three  projecting  blocks  provided 
with  strong  wooden  buttons  for  clamping  the  foot,  and 
there  is  a  round  hole  in  the  centre  of  the  stand  st,  so  that 
the  apparatus  can,  if  desired,  be  used  in  the  horizontal 
position,  the  light  being  sent  directly  through  the  hole. 
It  is  well  to  unscrew  and  remove  the  whole  of  the  upper 
part  of  the  microscope  tube  (Fig.  1,  £'),  the  cloth-lined 
tube  at  the  lower  end  of  the  camera  being  made  to  fit 
over  the  lower  and  larger  part  (t). 

When  the  apparatus  is  in  the  position  shown  in  the 
figure,  the  ground-glass  camera  plate  is  horizontal,  and  it 
would  be  extremely  awkward  to  lean  over  and  observe 
the  image  upon  the  plate  and  at  the  same  time  adjust  the 
focus.  To  obviate  this  difficulty  the  plane  mirror  m  is 
provided;  it  is  inclined  at  a  convenient  angle,  so  that  the 
image  on  the  plate  is  reflected  towards  the  observer  stand- 
ing behind  the  microscope.  When  focussing,  the  upper 
part  of  the  apparatus  as  well  as  the  head  of  the  observer 
may  be  enveloped  in  a  loose  black  cloth. 

It  is  necessary  to  employ  bright  sunlight  (or  some  other 
intense  and  actinic  source  of  light),  which  should  be 
allowed  to  pass  through  a  glass  vessel  containing  a  solu- 
tion of  amrnonio-sulphate  of  copper,  so  as  to  render  the 
light  mono-chromatic.  It  is  then  received  on  the  mirror 
of  the  microscope  (supposing  the  apparatus  to  be  vertical) 
either  directly  or  after  traversing  a  condensing  lens,  and 
is  reflected  by  the  mirror  up  through  the  object  and  the 
tube  of  the  microscope  in  the  ordinary  way.  An  objective 
of  any  magnifying  power,  even  an  immersion  may  be  em- 
ployed, provided  there  be  light  enough'.  With  the  same 
objective  greater  or  less  magnification  is  obtained  accord- 
ing as  the  camera  is  raised  or  lowered. 

The  object  is  focussed  upon  the  ground-glass  with  the 
utmost  exactitude.  The  light  is  then  cut  off,  the  mirror, 
w,  removed,  and  the  sensitized  plate  substituted  for  the 
ground-glass  plate  as  in  the  ordinary  process. 

Mr.  G.  Giles  has  devised   a  simple  mode  of  adapting 


APPENDIX.  297 

an  ordinary  photographic  camera  to  the  microscope,  of 
which  a  brief  description  will  be  found  in  the  u  Quarterly 
Journal  of  Microscopical  Science,"  1876,  p.  111.  It  ap- 
pears to  have  yielded  good  results,  but  for  wet  prepara- 
tions has  the  disadvantage  that  the  microscope  must  be 
placed  horizontally. 

Employment  of  Eosin  as  a  staining  fluid,— 
Dilute  solutions  of  eosin,  an  aniline-preparation  newly 
introduced  into  commerce,  have  recently  been  much  re- 
commended for  coloring  the  tissues.  The  dye  can  be 
used  dissolved  either  in  water  or  alcohol.  For  the  watery 
solution  Dreschfeld  recommends  a  strength  of  about 
1  per  1000;  this  takes  from  a  minute  to  a  minute  and 
a-halfto  stain  sections;  they  are  subsequently  put  for 
a  very  short  time  into  water  slightly  acidulated  with 
acetic  acid,  and  then  either  examined  in  glycerine  or 
mounted  in  dammar.  For  portions  of  tissue  which  are 
€to  be  hardened  in  alcohol,  the  process  of  hardening  and 
staining  can  be  effected  simultaneously  by  the  employ- 
ment of  an  alcoholic  solution  of  eosin.  The  color  im- 
parted by  eosin  is  a  rose-red.  (E.  Fischer,  u  Arch.  f.  Micr. 
Anat."  1875,  p.  349  ;  J.  D resell leld,  "Journal  of  Anatomy 
and  Physiology,"  Oct.  1876.) 


INDEX. 


A  BSORPTION  of  fat,  218 
XI     Adipose  tissue,  87 

development  of,  88 
Alkanet  for    injecting    lymphatics, 

177 

Ammonia  bichromate,    for   harden- 
ing nervous  tissue,  243 
Aniline  blue-bl;ick,      for      staining 

nervous  tissue,  244 
blue,  for  staining  peptic  cells, 

214 
for     staining    cerebellum, 

244 

Appliances  for  microscopic  work,  19 
Areolar  tissue,  action  of  acetic  acid 

on,  70 
corpuscles,  71 

stained  with  logwood, 

73 

constrictions  on  fibre  bun- 
dles, 74 
elastic  fibres,  71 

stained  with   ma- 
genta, 76 
fibres  of,  70 
interstitial      injection      of 

gelatine  into,  76 
preparation  by  method   of 

localized  oedema,  77 
prepared   with    nitrate    of 

silver,  80 

Arytenoid  cartilage  of  ox,  97 
Axis-cylinders  in  spinal  cord,  130 


BERLIN-BLUE,  mode  of  prepar- 
ing, 164 

reduction  of,  in  tissues,  170 
solution  for  injecting  lym- 
phatics, 177 
Blood,  action  of  electric  shocks  on, 

51 

human,  action  of  reagents  on, 
36 


Blood,  human,  action  of — 
acetic  acid,  38 
alkalies,  39 
chloroform,  39 
tannic  acid,  38 
water,  37 

mode  of  obtaining,  25 
on  warm  stage,  30 

effect     of     super- 
heating, 36 

Blood  corpuscles,  human,  red,  ap- 
pearance varies  with 
distances  of  objec- 
tive, 27 

effect  of  salt  upon,  28 
observation  of,  26 
structure  of,  36 
mode  of  counting,  289 
human,  white.  28 

amoeboid     movements 

of,  32 

development  of,  88 
of  frog,  42 

action  of  reagents  on,  36 

iodine,  52 

feeding    of    white    corpus- 
cles, 43 

migration  of  white  corpus- 
cles, 45 
influence    of    warmth    on 

white  corpuscles,  49 
of  newt,  action  of  boracic  acid, 

48 

action  of  carbonic  acid,  48 
Blood  crystals,  53 
Bloodvessels,  development  of,  88 
injection  of,  162 
larger,  epithelioid      lining     of, 
shown     by     nitrate      of 
silver,  138 

elastic  layers  of,  140 
fenestrated    membrane    of, 

140 
muscular  tissue  of,  140 


300 


INDEX. 


Bloodvessel?,  larger — 

mode  of  hardening,  141 
sections  of,  141 
sub-epithelial  layer  of,  139 
smaller,  epithelioid  cells  of,  150 
muscular      structure     and 

nuclei,  152 

Bone,  corpuscles  in  lacunae,  104 
hard,  grinding  section  of,   100 
precautions    in    mounting, 

101 
softened  in  hydrochloric  acid, 

102 

in  chromic  acid,  102 
in  picric  acid,  104 
lamellae      and      Sharpey's 

fibres,  105 
Brownian  movement,  35 


CABINET  for  keeping  specimens 
\J     in,  23 

Camera  lucida  of  Zeiss,  for  deline- 
ating objects,  288 

Canada  balsam,  solution  in  chloro- 
form, 149 

Canulas  for  injecting;   mode  of  pre- 
paring, 168 

Capillary  tubes,  mode  of  making.  43 
Carmine  gelatine  injection,  162 

solution  for  staining  tissues,  207 
for  staining  sections  of  sto- 
mach, 214 

Cartilage,  articular,  in  fresh  state,  89 
vertical      and      tangential 

sections  of,  92 
cell  spaces  of,  93 
Cartilage-cells,  action  of  water  on, 

89 

preservation  of,  91 
stained  by  chloride  of  gold,  94 
Cartilage,  costal,  96 

matrix,  cell  territories  of,  97 

stained  by  logwood,  97 
transition  between  hyaline  and 

yellow,  97 

Cell  spaces  of  connective  tissue,  78 
Central   tendon   of  diaphragm  pre- 
pared with  nitrate  of  silver,  175 
Cerebellum,  243 
Cerebrum,  243 
Chloral  hydrate  for  preparing  retina, 

267 
Choj-oid  coat  of  eye,  260 

bloodve.-sels  of,  273 
lamina     suprachoroi- 
dea,  261 


Choroid  coat  of  eye — 

layers  of,  262 
Ciliary   motion,   action   of  reagents 

on,  65 

carbonic  acid  on,  66 
chloroform  on,  66 
warmth  on,  65 
weak  alkalies,  65 
Ciliary  muscle,  261 
Circulation  in  otnentum  of  guinea- 
pig,   157 

in  frog's  web,  154 
in  lung  of  toad,  158 
in  mesentery  of  toad,  155 
in  tongue  of  toad,  158 
Cochlea,  mode  of  procuring,  277 
precautions  for  embedding  and 

cutting,  278 
softening    of  osseous    parietes, 

278 

teased  preparations  of,  279 
Cohnheim's  gold  method,  94 
Condenser,  bull's-eye,  use  of,   15 

used    as    dissecting    lens, 

17 

Conjunctiva,  bloodvessels  of,  272 
Connective-tissue      corpuscles       in 

areolar  tissue,  71 
tongue  of  toad,   159 
Cornea,  cell  spaces  of,  257 

mode  of  injecting,  257 
epithelium  of,  251 
mode  of  hardening,  250 
precautions  to  avoid  curling  up 

of  sections,  250 
substantia  propria  of,  251 
of  frog,  corputcles   and   nerves 

of,  252 
of  rabbit,  corpuscles  and  nerves 

of,  254 

isolation  of  corpuscles,  255 
nerves  of,  256 

Corneal  tubes  of  Bowman,  259 
Corneo-sclerotic  junction,  259 
Covei -glasses,  mode  of  averting 

pressure  of,  42 
mode  of  cleaning,  19 
fixing,  61 

by  paraffin,  106 


DAMMAR  varnish,  149 
Delineation  of  microscopic  ob- 
jects, 238 
Diaphragm  of  microscope,    use    of, 

13 
Directions  for  work,  23 


INDEX. 


301 


ELASTIC  fibres  in  areolar  tissue,  ' 
71 

transverse  section  of,  82 
networks  of  serous  membrane, 

82 

of  artery,  140 
tissue,  82 

Electricity,  mode  of  applying,  50 
Embedding  in  wax-mass,  142 
moulds  for,  143 
by  gum  method,  190 
membranous    viscera,    precau- 
tions for.  212 
Endocardium.  194 

fibres  of  Purkinje  in,  195 
End-plates  of  mammals,  135 

of  lizard,  136 
Eosin,  as  a  staining  fluid,  297 

for    staining     nervous    tissue, 

244 
Epidermis,     cells    of    horny    layer  j 

shown  by  potash,  58 
Epithelioid-cells,  78 

covering  tendon,  87 
Epithelium,    ciliated,    from     frog's  > 

mouth,  62 

from  gills  of  mussel,  63 
study    of    separated   cells,  j 

78 

columnar,  59 
scaly,  of  mouth,  56 

deeper  layers  of,  57 
Erectile  tissue,  mode  of  hardening, 

237  . 
Eye,  bloodvessels  of,  272 

general  mode  of  preparing,  246 
of  pig  as  a  substitute  for  human 

eye,  247 

Eyelids,  sections  of,  247 
Eye-piece  of  microscope,  15 


FAT-CELLS,  87 
membrane  of,  88 
development  of,  88 
Fibrine  in  blood,  30 
Fibro-cartilage,  yellow,  of  epiglot- 
tis, 98 

transition  to  hyaline,  97 
white,  98 
Fibrous  tissue,  83 
Field  glass,  15 
Forceps,  20 
Freezing    method    of    hardening — 

Dr.  Rutherford's,  293 
Dr.     Urban     Pritchard's, 

123 
26 


GANGLIA,  sections  of,  192 
Ganglion  cells,  131 
Gas,  carbonic  ncid,  mode  of  apply- 
ing to  a  preparation,  49 
chamber,  40 
Gastric  glands,  213 

cells  of  isolated,  215 
Gelatine  injecting  fluid,  162 

mode    of     preserving, 

171 
Glycogen,     its    presence    in    white 

blood  corpuscles,  52 
Goblet  cells,  60 
Gold  chloride,   method  of  staining 

with,  94 
Granules,  masses  of,  in  blood,  29 

Dr.     Osier's     observa- 
tions upon,  33 
Gullet.     See  (Esophagus. 


TJ^MATOXYLIN.   See  Logwood. 
JLl     Haemin  crystals,  54 
Haemoglobin  crystals,  53 
Hairs,  188 

development  of,  207 
Hayem  and  Nachet's-  mode  of  count- 
ing blood  corpuscles,  290 
Heart,  muscular  substance  of,  193 

lymphatics  of,  195 
Hepatic  cells,  228 


IMMERSION  objectives,  mode  of 

JL     using,  16 

Inflammation  changes  in  mesentery 

of  toad,  157 
in  ti  ngue  of  toad,  161 
Injected  parts,  mode  of  preparing, 

169 
Injection  apparatus,  165 

of  an  entire  animal,  167 
of  bloodvessels,   162 

mode  of  killing  an  animal 

for,  225 

of  lymphatics,  177 
mass,  mode  of  preparing,  162 
Injections  fluid  in  the  cold,  170 
Instruments    required     for    micro- 
scopic preparation,  20 
Intestine,  large,  222 

small,  bloodvessels  of,  219 
mode  of  hardening,  217 
nerves  of,  219 
precautions  for  embedding, 

217 
Iodized  serum,  59 


302 


INDEX. 


Iris,  bloodvessels  of.  273 
muscular  tissue  of,  263 
sections  of,  259 


KIDNEY,  bloodvessels  of,  233 
examination   in   fresh  con- 
dition, 234 

mode  of  hardening,  231 
tubules,  isolation   of,  by   Lud- 

wig's  method,  233 
epithelium  of,  232 
basement    membrane   of, 
stained   with   nitrate  of 
silver,  234 

transverse  section  of,  232 
Klein,    Dr.    E.,    on    structure    of 
spleen,  230 


LABIA,  mode  of  preparing,  238 
Lachrymal  gland,  248 
Larynx,  201 

Lens,  isolation  of  fibres,  269 
sections  of,  270 
suspensory  ligament  of,  272 
capsule,  epithelium  of,  271 
Liver,  bloodvessels  of,  223 
lymphatics  of,  227 
mode  of  hardening,  223 

of  injecting  bile-ducts,  226 
Logwood  solution  for  staining  tis- 
sues, 22 

Kleinenberg's,  198 
Lung,  embedding   by  cacao-butter 

process,  198 

epithelium  of  air-cells,  199 
injection  of  bloodvessels,  200 
mode  of  hardening,  197 
Lymphatic  glands,  184 
Lymphatics,  injection  of,  177 

of  diaphragm,   natural    injec- 
tion of,  181 
of  tendon,  mode  of  injecting, 

179 

larger,  177 

smaller,  in  omentum  and  cen- 
tral tendon,  172-5 


MAGNIFYING   power  of  micro- 
scope, estimation  of,  286 
Malassez's  mode  of  counting  blood 

corpuscles,  290 
Mammary  glands,  242 
Marrow,  red,  on  warm  stage,  110 
giant  cells  of,  111 


1  Measurement   of  an    object   under 

the  microscope,  285 
Mesentery,  stained  with  nitrate  of 

silver,  151 
circulation  in,  155 
Micrometers,  285 
Microphotography,  294 
Microscope,  biuoc»lar,  use  of,  15 
best  test-objects  for,  17 
eye-piece  of,  15 
for  dissection,  17 
parts  of,  13 
powers  of,  16 

selection  of,  by  student,  17 
Microtome,  Stirling's,  291 
Ranvier's,  292 
Rutherford's,  293 
Migration  of  white  corpuscles  from 

veins,  161 

Moist  chamber  made  with  putty,  39 
Mouth,  mucous  membrane  of,  204 
Mucous  glands  of  tongue,  207 
Muscle,  bloodvessels  of,  125 

ending   in   tendon    in   mouse's 

tail,  124 
endingin  tendon  in  frog's  neck, 

124 
examined    by  polarized   light, 

120 

section  of,  in  frozen  state,  123 
transverse  section  of,  122 
of  water-beetle,  118 

mode  of  production  of 
the     transverse 
striae,  119 
studying  contrac- 
tion of,  119 
involuntary,  mode  of  isolating 

cells  of,  112 
showing  nuclei,  115 
prepared   with    nitrate    of 

silver,  113 
voluntary,  action  of  acetic  acid, 

115 
demonstration    of     sarco- 

lemma  in,  115 
mammalian,  114 
isolation  of  fibres,  116 
separation    of,  into    disks 

and  fibrils,  116 
Muscle-rods,  118 


N 


AIL,  cells  of,  separated  by  pot- 
ash, 58 

embedded  by  gum  method,  190 
sections  of,  189 


INDEX. 


303 


Needles,  mounted,  20 
Nerve  cells  of  ganglia,  131 

of  spinal  cord,  130 
Nerve  fibres,  medullated,  126 

in  spinal  cord,  130 
treated     with      osmic 

acid,  129 
stained  with  nitrate  of 

silver,  129 
non-medullated,  127 
Nerve  trunk,  perineurium  of,  shown 

by  silver  method,  128 
sections  of,  191 
structure  of,  128 
Nerves,  motor,  ending  of,  135 
Nervous  plexuses  of  intestine,  219, 

222 

Nervous   system,  central,  mode   of 
preparing,  243 


OBJECT-GLASS,  or  objective,  15 
Objective,  immersion,  16 
Ocular  of  microscope,  15 
(Esophagus,  211 

bloodvessels  of,  212 
Oil  of  cloves  for  clarifying  sections, 

148 

Oil-globules,  effect  on  light,  119 
Olfactory  mucous  membrane,  281 
Ornentum,  prepared  with  nitrate  of 

silver,  172 
circulation  in,  157 
Optical  section,  153 
Organ  of  taste,  282 
Osmic  acid  colors  fatty  substances 

black,  61 
for  nerve,  129 
for  retina,  264-66 

Ossification,  intracartilaginous,  107 
intramembranous,  106 
of  lower  jaw,  206 
Ovary,  238 

Ovum,  mode  of  obtaining,  239 
impregnation  of,  240 


PACINIAN  corpuscles  from  cat's 
mesentery,  132 
sections  of,  135 
treatment    with     chromic 

acid,  134 

nitrate  of  silver,  134 
osmic  acid,  133 
Palate,  209 
Pericardium,  193 
Perineurium,  lymph  spaces  of,  191 


Pia  mater,  vessels  of,  152 
Picrocarminate  of  ammonia,  85 
Pigment,  hexagonal,  of  retina,  268 
Pipettes,  mode  of  making,  22 
Pleura,  196 

Polarization  apparatus,  17 
Polarizing  microscope,  120 
Pritchard's,  Dr.  Urban,  mode  of 

freezing  tissues,  123 
Prostate  gland,  237 
Pulmonary  vessels,  injection  of,  200 
Purkinje,  fibres  of,  195 


"REAGENTS  in  common  use,  22 
XX        action  of,   upon   the  blood, 

36 

bottles  for,  22 
mode  of  applying,  36 
Recklinghausen's  silver  method,  78 
Remak,  fibres  of,  127 
Retina,  bloodvessels  of,  274 
fibres  of  Miiller,  265,  267 
fresh,  in  vitreous  humor,  267 
in  iodized  serum,  268 
isolation  of  elements,  266 
methods  of  hardening  and  cut- 
ting sections,  264 
of  bird,  reptile,  and  fish,  269 
of  frog,  268 


SALIVARY  corpuscles,  57 
glands,  209 
Salt  solution,  22 
Sankey's,  Mr.  H.  R.  0.,  method  of 

preparing  sections  of  brain,  244 
Sclerotic  coat  of  eye,  248 

bloodvessels  of,  272. 
lamina  fusca,  249 
Scissors,  20 
Scrotum,   238 
Section -lifter,  148 
Sections,  mode  of  cutting,  145 
staining,  147 

with        Kleinenberg's 

logwood,  149 
mounting,    149 
Semicircular  canals,  mode  of  finding 

and  preparing,  275,  276 
Seminiferous     tubules,     epithelioid 

cells  of,  241 
isolation  of,  241 
Silver  nitrate,   method  of  staining 

with,  78 
Skin,  185 

bloodvessels  of,  187 


30  i 


INDEX. 


Skin- 
double  staining  of,  186 
preparation  of,  by  Dr.  W.  Stir- 

ling's  method,  187 
Slides  for  microscopic  purposes,  19 
Spinal  cord.  243 

isolation  of  cells  of,  129 
Spleen,  229 

demonstration  of  retiform  tissue 

of,   230 

injection  of,  230 
Stomach,  212 

bloodvessels  of,  215 
glands  of,  213 
horizontal  sections  of,  214 
lymphatics  of,  215 
Stomata    in    lymphatic    septum    of 

frog,   176 

Suprarenal  capsule,  235 
Synovial  membranes,  182 

bloodvessels  of,  183 
Haversian  fringes  of,  183 


TACTILE  corpuscles,  186 
Tarsal  cartilage  of  eyelid,  248 
Taste-buds  in  papillae  foliatae  of  rab- 
bit,  282 

circumvallatae,  208 
Teeth,  in  situ,  206 

development  of,  206 
dentinal  sheaths  of,  205 
sections  obtained  by  grinding, 

204 

sections  of,  softened,  204 
Teeth,  soft  tissues  of,  205 
Tendon  of  mouse's  tail,  83 

action   of    acetic    acid 

on,  84 

cell  spaces  of,  85 
transverse  section,  86 
cells,  85 


i  Tenon,  capsule  of,  248 
Testis,  240 
Thymus  gland,  202 
Thyroid  body,  202 
Tongue,  207 

bloodvessels  of,  208 

of  toad,  158 
Tonsils,  209 
Trachea,  201 

epithelium  of,  67 
Tunica  vaginalis,  241 


DRETERS,  235 
Urinary  bladder,  236 
Uriniferous  tubules.     See  Kidney. 
Uterus,  238 


VAGINA,  238 
Vaginal  synovial  membranes, 
182 

Vesiculae  seminales,  237 
Vitreous  humor,  hyaloid  membrane 
of,  271 


WARMING  apparatus,    with  gas 
regulator,  for  chloride  of  gold 
preparations,  96 
Warm  stage,  simple,  30 

mode      of    estimating 

temperature,  31 
with  gas  regulator,  34 
Water,  action  of,  on  fresh  tissues, 

37 
Wax-mass  for  embedding,  142 


70NULE  of  Zinn,  71 

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8vo.  vol.  of  over  1000  pages,  containing  213  illustrations.     Cloth,  $4. 

pauRCHiLL  (FLEETWOOD).   ON  THE  THEORY  AND  PRACTICE 

U  OF  MIDWIFERY.  With  notes  and  additions  by  D.  Francis  Condie, 
M.D.  With  about  200  illustrations.  In  one  handsome  8vo.  vol.  of 
nearly  700  pages.  Cloth,  $4  ;  leather,  $5. 

ESSAYS  ON  THE  PUERPERAL  FEVER,  AND  OTHER  DIS- 
EASES PECULIAR  TO  WOMEN.  In  one  neat  octavo  vol.  of  about 
450  pages.  Cloth,  $2  50. 

C3NDIE  (D.  FRANCIS).     A  PRACTICAL  TREATISE  ON  THE  DIS- 
EASES OF  CHILDREN.     Sixth  edition,  revised  and  enlarged.     In 
one  large  8vo.  vol.  of  800  pages.     Cloth,  $5  25  ;  leather,  $6  25. 
pLOWES   (FBANK).     AN   ELEMENTARY   TREAT'ESE   ON  PRAC  - 
V     TICAL  CHEMISTRY  AND  QUALITATIVE  INORGANIC  ANA- 
LYSIS.    Especially  adapted  for  Lab^ratary  Use.    From  the  Second 
English  Edition.     In  one  royal  12oio.  vol.     Cloth,  $2  50.     (Just 
issued.) 


HENRY  C.  LEA'S  PUBLICATIONS. 


D 
D 


nULLERIER  (A.)     AN  ATLAS  OF  VENEREAL  DISEASES.     Trans- 
*J     lated  and  edited  by  FREEMAN  J.  BUMSTEAD,  M.D.     A  large  imperial 
quarto  volume,  with  26  plates  containing  about  150  figures,  beauti- 
fully colored,  many  of  them  the  size  of  life.     In  one  vol.,  strongly 
bound  in  cloth,  $17. 

Same  work,  in  five  parts,  paper  covers,  for  mailing,  $3  per  part. 

pYCLOPEDIA  OF  PRACTICAL  MEDICINE.     By  Dunglison,  Forbes, 
^     Tweedie,  and  Conolly.     In  four  large  super-royal  octavo  volumes,  of 

3254  double-columned  pages,  leather,  raised  bands,  $15.  Cloth,  $11. 
CAMPBELL'S  LIVES  OF  LORDS  KENYON,  ELLENBOROUGH,  AND 
\J  TENTERDEN.  Being  the  third  volume  of  "  Campbell's  Lives  of 

the  Chief  Justices  of  England."   In  one  crown  octavo  vol.   Cloth,  $2. 

D ALTON  (J.  C.)  A  TREATISE  ON  HUMAN  PHYSIOLOGY.  Sixth 
edition,  thoroughly  revised,  and  greatly  enlarged  and  improved,  with 
316  illustrations.  In  one  very  handsome  8vo.  vol.  of  830  pp. 
Cloth,  $5  50  ;  leather,  $6  50.  (Just  issued.) 

AVIS  (F.  H.)  LECTURES  ON  CLINICAL  MEDICINE.  Second 
edition,  revised  and  enlarged.  In  one  12mo.  vol.  Cloth,  $1  75. 

ON  QUIXOTE  DE  LA  MANCHA.  Illustrated  edition.  In  two  hand- 
some vols.  crown  8vo.  Cloth,  $2  50  ;  half  morocco,  $3  70. 

DEWEES  (W.  P.)  A  TREATISE  ON  THE  DISEASES  OF  FEMALES. 
With  illustrations.  In  one  8vo.  vol.  of  536  pages.  Cloth,  $3. 
DRUITT  (ROBERT).  THE  PRINCIPLES  AND  PRACTICE  OF  MO 
DERN  SURGERY.  A  revised  American,  from  the  eighth  London 
edition.  Illustrated  with  432  wood  engravings.  In  one  8vo.  vol. 
of  nearly  700  pages.  Cloth,  $4  ;  leather,  $5. 

DTTNGLISON  (ROBLEY).  MEDICAL  LEXICON;  a  Dictionary  of 
Medical  Science.  Containing  a  concise  explanation  of  the  various 
subjects  and  terms  of  Anatomy,  Physiology,  Pathology,  Hygiene, 
Therapeutics,  Pharmacology,  Pharmacy,  Surgery,  Obstetrics,  Medical 
Jurisprudence,  and  Dentistry.  Notices  of  Climate  and  of  Mineral 
Waters;  Formulae  for  Officinal,  Empirical,  and  Dietetic  Preparations, 
with  the  accentuation  and  Etymology  of  the  Terms,  and  the  French 
and  other  Synonymes.  In  one  very  large  royal  8vo.  vol.  New  edi- 
tion. Cloth,  $6  50;  leather,  $7  50.  (Just  issued.) 

HUMAN   PHYSIOLOGY.     Eighth   edition,   thoroughly  revised. 

In  two  large  8vo.  vols.  of  about  1500  pp.,  with  532  illus.     Cloth,  $7. 

DE  LA  BECHE'S  GEOLOGICAL  OBSERVER.  In  one  large  8vo.  vol. 
of  700  pages,  with  300  illustrations.  Cloth,  $4. 

DANA  (JAMES  D.)  THE  STRUCTURE  AND  CLASSIFICATION  OF 
ZOOPHYTES.  With  illust.  on  wood.  In  one  imp.  4to.  vol.  Cloth,  $4. 
ELLIS  (BENJAMIN).  THE  MEDICAL  FORMULARY.  Being  a 
collection  of  prescriptions  derived  from  the  writings  and  practice  of 
the  most  eminent  physicians  of  America  and  Europe.  Twelfth  edi- 
tion, carefully  revised  by  A.  H.  Smith,  M.  D.  In  one  8vo.  volume 
of  374  pages.  Cloth,  $3. 

TiRICHSEN   (JOHN).     THE    SCIENCE   AND   ART   OF   SURGERY. 
JJ     A  new  and  improved   American,  from  the  sixth  enlarged  and  re- 
vised London  edition.    Illustrated  with  630  engravings  on  wood.    In 
two  large  8vo.  vols.     Cloth,  $9  00 ;  leather,  raised  bands,  $11  00. 
•pNCYCLOPJEDIA  OF  GEOGRAPHY.     In  three  large  8vo.  vols.     Illus- 
-*-1     trated  with  83'maps  and  about  1100  wood-cuts.     Cloth,  $5. 

FOTHERGILL'S  PRACTITIONER'S  HANDBOOK  OF  TREATMENT.  . 
In  one  handsome  8vo.  vol.  of  about  550  pp.  Cloth,  $4.  (Just  issued.) 


HENRY  C.  LEA'S  PUBLICATIONS. 


pARQUHARSON    (ROBERT).        A    GUIDE    TO    THERAPEUTICS. 
•L      Edited,    with   additions,    embracing  the   U.    S.  Pharmacopoeia,   by 

Frank  Woodbury,  M.D.     In  one  neat  royal  12mo.  volume.     Cloth. 

(Nearly  ready.) 

FENWICK  (SAMUEL).  THE  STUDENTS'  GUIDE  TO  MEDICAL 
DIAGNOSIS.  From  the  Third  Revised  and  Enlarged  London  Edi- 
tion. In  one  vol.  royal  12mo.  Cloth,  $2  25. 

FLETCHER'S  NOTES  FROM  NINEVEH,  AND  TRAVELS  IN  MESO. 
POTAMIA,  ASSYRIA,  AND  SYRIA.  In  one  12mo.  vol.  Cloth,  75  cts. 
TPOX  (TILBURY).     EPITOME  OF  SKIN  DISEASES,  with  Formula 
•t     for  Students  and  Practitioners.    In  one  small  12ino.  vol.    Cloth,  $1. 
pLINT    (AUSTIN).     A    TREATISE    ON    THE    PRINCIPLES    AND 
J-      PRACTICE  OF  MEDICINE.     Fourth  edition,  thoroughly  revised 
and  enlarged.     In  one  large  8vo.  volume  of  1070  pages.     Cloth,  $6  ; 
leather,  raised  bands,  $7.     (Just  issued.) 

A  PRACTICAL  TREATISE  ON  THE  PHYSICAL  EXPLORA- 
TION OF  THE  CHEST,  AND  THE  DIAGNOSIS  OF  DISEASES 
AFFECTING  THE  RESPIRATORY  ORGANS.    Second  and  revised 
edition.     One  8vo.  vol.  of  595  pages.     Cloth,  $4  50. 

A  PRACTICALTREATISEONTHE  DIAGNOSIS  ANDTREAT 

MENT  OF  DISEASES  OF  THE  HEART.  Second  edition,  enlarged 
In  one  neat  8vo.  vol.  of  over  500  pages,  $4  00. 

ON  PHTHISIS  :  ITS  MORBID  ANATOMY,  ETIOLOGY,  ETC. 

in  a  series  of  Clinical  Lectures.    A  new  work.    In  one  handsome  8vo. 
volume.     Cloth,  $3  50.     (Just  issued.) 

A  MANUAL  OF  PERCUSSION  AND  AUSCULTATION;  of  the 

Physical  Diagnosis  of  Diseases  of  the  Lungs  and  Heart,  and  of  Tho- 
racic Aneurism.  In  one  handsome  royal  12mo.  volume.  Cloth, 
$1  75.  (Just  issued.) 

MEDICAL  ESSAYS.     In  one  neat  12mo.  volume.     Cloth,  $1  38. 

pOWNES  (GEORGE).  A  MANUAL  OF  ELEMENTARY  CHEMISTRY. 
•t      From  the  tenth  enlarged  English  edition.     In  one  royal  12mo.  vol.  of 

857  pages,  with  197  illustrations.     Cloth,  $2  75  ;  leather,  $3  25. 

FULLER  (HENRY).  ON  DISEASES  OF  THE  LUNGS  AND  AIR 
PASSAGES.  Their  Pathology,  Physical  Diagnosis,  Symptoms,  and 
Treatment.  From  the  second  English  edition.  In  one  8vo.  vol. 
of  about  500  pages.  Cloth,  $3  50. 

GALLOWAY  (ROBERT).  A  MANUAL  OF  QUALITATIVE  AN- 
ALYSIS. From  the  fifth  Eng.  ed.  In  one  12mo.  vol.  Cloth,  $2  60, 
GLTJGE  (GOTTLIEB).  ATLAS  OF  PATHOLOGICAL  HISTOLOGY. 
Translated  by  Joseph  Leidy,  M.D.,  Professor  of  Anatomy  in  the 
University  of  Pennsylvania,  &c.  In  one  vol.  imperial  quarto,  with 
320  copperplate  figures,  plain  and  colored.  Cloth,  $4. 

GREEN  (T.  HENRY).  AN  INTRODUCTION  TO  PATHOLOGY  AND 
MORBID  ANATOMY.  Second  Amer.,  from  the  third  Lond.  Ed. 
In  one  handsome  8vo.  vol.,  with  numerous  illustrations.  Cloth, 
$2  75.  (Just  issued  ) 

GRAY  (HENRY).  ANATOMY,  DESCRIPTIVE  AND  SURGICAL. 
A  new  American,  from  the  fifth  and  enlarged  London  edition.  In  on* 
large  imperial  8vo.  vol.  of  about  900  pages,  with  462  large  and  elabo- 
rate engravings  on  wood.  Cloth,  $6;  leather,  $7.  (Lately  issued.) 
GRIFFITH  (ROBERT  E.)  A  UNIVERSAL  FORMULARY,  CON- 
TAINING THE  METHODS  OF  PREPARING  AND  ADMINISTER-' 
ING  OFFICINAL  AND  OTHER  MEDICINES.  Third  and  Enlarged 
edition.  Edited  by  John  M.  Maisch.  In  one  large  8vo  vol  of  800 
pages,  double  columns.  Cloth,  $4  50  ;  leather,  $5  50. 


HENRY  C.  LEA'S  PUBLICATIONS. 


GROSS  (SAMUEL  D.)  A  SYSTEM  OF  SURGERY,  PATHOLOGICAL, 
DIAGNOSTIC,  THERAPEUTIC,  AND  OPERATIVE.  Illustrated 
by  1403  engravings.  Fifth  edition,  revised  and  improved.  In  two 
large  imperial  8vo.  vols.  of  over  2200  pages,  strongly  bound  in 
leather,  raised  bands,  $15. 

(VROSS  (SAMUEL  D.)  A  PRACTICAL  TREATISE  ON  THE  DIS- 
™  eases,  Injuries,  and  Malformations  of  the  Urinary  Bladder,  the  Pros- 
tate Gland,  and  the  Urethra.  Third  Edition,  thoroughly  Revised 
and  Condensed,  by  Samuel  W.  Gross,  M.D.,  Surgeon  to  the  Phila- 
delphia Hospital.  In  one  handsome  octavo  volume,  with  about  two 
hundred  illustrations.  Cloth,  $4  50.  (Just  issued.) 

A  PRACTICAL  TREATISE  ON  FOREIGN  BODIES  IN  THE 

AIR  PASSAGES.    In  one  8vo.  vol.  of  468  pages.    Cloth,  $2  75. 

GIBSON'S  INSTITUTES  AND  PRACTICE  OF  SURGERY.  In  two  8vo. 
vols.  of  about  1000  pages,  leather,  $6  50. 

GOSSELIN  (L  )  CLINICAL  LECTURES  ON  SURGERY.  Delivered 
at  the  Hospital  of  La  Charite  Translated  from  the  French  by  Lewis 
A.  Stiimon,  M.D.,  Surgeon  to  the  Presbyterian  Hospital,  New  York. 
With  illustrations.  (Publishing  in  the  Medical  News  and  Library 
for  1876-7.) 

HUDSON  (A.)  LECTURES  ON  THE  STUDY  OF  FEVER.  1  vol. 
8vo.,  316  pages.  Cloth,  $2  50. 

HEATH  (CHRISTOPHER).  PRACTICAL  ANATOMY  ;  A  MANUAL 
OF  DISSECTIONS.  With  additions,  by  W.  W.  Keen,  M.  D.  In  1 
volume  ;  with  247  illustrations.  Cloth,  $3  50  ;  leather,  $4. 

HARTSHORNE  (HENRY).  ESSENTIALS  OF  THE  PRINCIPLES 
AND  PRACTICE  OF  MEDICINE.  Fourth  and  revised  edition. 
In  one  12ino.  vol.  Cloth,  $263;  half  bound,  $288.  (Lately  issued  ) 

CONSPECTUS  OF  THE  MEDICAL   SCIENCES.      Comprising 

Manuals  of  Anatomy,  Physiology,  Chemistry,  Materia  Medica,  Prac- 
tice of  Medicine,  Surgery,  and  Obstetrics.     Second  Edition.     In  one 
royal   12mo.    volume  of  over   1000   pages,   with   477   illustrations. 
Strongly  bound  in  leather,  $5  00  ;  cloth,  $4  25.     (Lately  issued.) 

A  HANDBOOK  OF  ANATOMY  AND  PHYSIOLOGY.     In  one 

neat  royal  12iuo.  volume,  with  many  illustrations.     Cloth,  $1  75. 

HAMILTON  (FRANK  H.)  A  PRACTICAL  TREATISE  ON  FRAC- 
TURES AND  DISLOCATIONS.  Fifth  edition,  carefully  revised. 
In  one  handsome  8vo.  vol.  of  830  pages,  with  344  illustrations.  Cloth, 
$5  75  ;  leather,  $5  75.  (Just  issued.) 

HOLMES  (TIMOTHY).  SURGERY,  ITS  PRINCIPLES  AND  PRAC- 
TICE. In  one  handsome  8vo.  volume  of  1000  pages,  with  411  illus- 
trations. Cloth,  $6;  leather,  with  raised  bands,  $7.  (Just  ready.) 
HOBLYN  (RICHARD  D.)  A  DICTIONARY  OF  THE  TERMS  USED 
IN  MEDICINE  AND  THE  COLLATERAL  SCIENCES.  In  one 
12mo.  volume,  of  over  500  double-columned  pages.  Cloth,  $1  50; 
leather,  $2. 

HODGE  (HUGH  L.)  ON  DISEASES  PECULIAR  TO  WOMEN,  IN. 
CLUDING  DISPLACEMENTS  OF  THE  UTERUS.  Second  and 
revised  edition.  In  one  8vo.  volume.  Cloth,  $4  50. 
THE  PRINCIPLES  AND  PRACTICE  OF  OBSTETRICS.  Illus- 
trated with  large  lithographic  plates  containing  159  figures  from 
original  photographs,  and  with  numerous  wood-cuts.  In  one  large 
quarto  vol.  of  550  double-columned  pages.  Strongly  bound  in  cloth, 
$14. 


HENRY  C.  LEA'S  PUBLICATIONS. 


H 


TJOLLAND  (SIR  HENRY).    MEDICAL  NOTES  AND  REFLECTIONS. 
•»••*•    From  the  third  English  edition.    In  one  8vo.  vol.  of  about  500  pages. 
Cloth,  $3  50. 

HODGES  (RICHARD  M.)  PRACTICAL  DISSECTIONS.  Second  edi- 
tion. In  one  neat  royal  12mo.  vol.,  half  bound,  $2. 

TTUGHES.      SCRIPTURE    GEOGRAPHY  AND  HISTORY,   with   12 
•"•  colored  maps.    In  1  vol.  12mo.     Cloth,  $1. 

HORNER  (WILLIAM  E.)  SPECIAL  ANATOMY  AND  HISTOLOGY. 
Eighth  edition,  revised  and  modified.  In  two  large  8vo.  vols.  of  over 
1000  pages,  containing  300  wood-cuts.  Cloth,  $6. 

ILL  (BERKELEY).  SYPHILIS  AND  LOCAL  CONTAGIOUS  DIS- 
ORDERS. In  one  8vo.  volume  of  467  pages.  Cloth,  $3  25. 

SILLIER  (THOMAS).  HAND-BOOK  OF  SKIN  DISEASES.  Second 
Edition.  In  one  neat  royal  12mo.  volume  of  about  300  pp.,  with  two 
plates.  Cloth,  $2  25. 

HALL  (MRS.  M.)  LIVES  OF  THE  QUEENS  OF  ENGLAND  BEFORE 
THE  NORMAN  CONQUEST.  In  one  handsome  8vo.  vol.  Cloth, 
$2  25;  crimson  cloth,  $2  50  ;  half  morocco,  $3. 

TONES  (C.  HANDFIELD).     CLINICAL  OBSERVATIONS  ON  FUNC- 
U      TIONAL  NERVOUS  DISORDERS.     Second  American  Edition.     In 
one  8vo.  vol.  of  348  pages.     Cloth,  $3  25. 

EIRKES  (WILLIAM  SENHOUSE).  A  MANUAL  OF  PHYSIOLOGY. 
A  new  American,  from  the  eighth  London  edition.  One  vol.,  with 
many  illus.,  12mo.  Cloth,  $3  25;  leather,  $3  75. 

KNAPP  (F.)  TECHNOLOGY ;  OR  CHEMISTRY,  APPLIED  TO  THE 
ARTS  AND  TO  MANUFACTURES,  with  American  additions,  by 

Prof.  Walter  R.  Johnson.  In  two  8vo.  vols.,  with  500  ill.  Cloth,  $6. 
•JTENNEDY'S  MEMOIRS  OF  THE  LIFE  OF  WILLIAM  .WIRT.  In 
J-*-  two  vols.  12mo.  Cloth,  $2. 

T  EA  (HENRY  C.)    SUPERSTITION  AND  FORCE ;  ESSAYS  ON  THE 
•Ll     WAGER  OF  LAW,  THE  WAGER  OF  BATTLE,  THE  ORDEAL, 

AND  TORTURE.    Second  edition,  revised.    In  one  handsome  royal 

12mo.  vol.,  $2  75. 
STUDIES  IN  CHURCH  HISTORY.     The  Rise  of  the  Temporal 

Power — Benefit  of  Clergy — Excommunication.      In   one   handsome 

12mo.  vol.  of  515  pp.     Cloth,  $2  75. 
AN  HISTORICAL   SKETCH   OF   SACERDOTAL  CELIBACY 


IN  THE  CHRISTIAN  CHURCH.     In  one  handsome  octavo  volume 
of  602  pages.     Cloth,  $3  75. 

LA  ROCHE  (R.)     YELLOW  FEVER.     In  two  8vo.  vols.  of  nearly  1500 
pages.     Cloth,  $7. 

PNEUMONIA.    In  one  8vo.  vol.  of  500  pages.     Cloth,  $3. 

T  INCOLN  (D.  F.)     ELECTRO-THERAPEUTICS.     A  Condensed  Man- 
•"    ual  of  Medical  Electricity.     In  one  neat  royal  12mo.  volume,  with 

illustrations.     Cloth,  $1  50.     (Just  issued.) 

TEISHMAN  (WILLIAM).     A  SYSTEM  OF   MIDWIFERY.     Includ- 
J-'     ing  the  Diseases  of  Pregnancy  and  the  Puerperal  State.      Second 
American,  from  the  Second  English  Edition.     With  additions,  by 
J.  S.  Parry,  M.D.     In  one  very  handsome  8vo.  vol.  of  800  pages  and 
200  illustrations.     Cloth,  $5  ;   leather,  $6.     (Just  issued.) 
T  AURENCE  (J.  Z.)   AND  MOON  (ROBERT  C.)     A   HANDY-BOOK 
J-I     OF  OPHTHALMIC  SURGERY.     Second  edition,  revised  by  Mr. 
Laurence.     With  numerous  illus.     In  one  Svo.  vol.     Cloth,  $2  75. 


8  HENRY  C.  LEA'S  PUBLICATIONS. 

T  EHMANN  (C.  G.)     PHYSIOLOGICAL  CHEMISTRY.    Translated  by 
-*-1     George  F.  Day,   M.  D.     With  plates,  and  nearly  200  illustrations. 

In  two  large  8vo.  vols.,  containing  1200  pages.     Cloth,  $6. 
A    MANUAL   OF   CHEMICAL   PHYSIOLOGY.     In   one   very 

handsome  8vo.  vol.  of  336  pages.     Cloth,  $2  25. 

LAWSON  (GEORGE).    INJURIES  OF  THE  EYE,  ORBIT,  AND  EYE- 
LIDS,  with  about  100  illustrations.     From  the  last  English  edition. 

In  one  handsome  8vo.  vol.     Cloth,  $3  50. 

T  UDLOW  (J.  L.)     A  MANUAL  OF  EXAMINATIONS  UPON  ANA- 
-LJ     TOMY,  PHYSIOLOGY,  SURGERY,  PRACTICE  OF  MEDICINE, 

OBSTETRICS,  MATERIA  MEDICA,  CHEMISTRY,  PHARMACY, 

AND  THERAPEUTICS.     To  which  is  added  a  Medical  Formulary. 

Third  edition.     In  one  royal  12mo.  vol.  of  over  800  pages.     Cloth, 

$3  25  ;  leather,  $3  75. 

T  YNCH  (W.  F.)     A  NARRATIVE  OF  THE  UNITED  STATES  EX- 
JJ     PEDITION  TO  THE  DEAD  SEA  AND  RIVER  JORDAN.     In  one 

large  octavo  vol.,  with  28  beautiful  plates  and  two  maps.    Cloth,  $3. 

Same  Work,  condensed  edition.    One  vol.  royal  12mo.    Cloth,  $1. 

T  EE  (HENRY)  ON  SYPHILIS.     In  one  8vo.  vol.     Cloth,  $2  25. 

T  YONS  (ROBERT  D.)     A  TREATISE  ON  FEVER.     In  one  neat  Svo. 
-Ll     vol.  of  362  pages.     Cloth,  $2  25. 

MARSHALL    (JOHN).      OUTLINES    OF    PHYSIOLOGY,    HUMAN 
AND  COMPARATIVE.     With  Additions  by  FRANCIS   G.  SMITH. 
M.  D.,  Professor  of  the  Institutes  of  Medicine  in  the  University  of 
Pennsylvania.     In  one  Svo.  volume  of  1026  pages,  with  122  illustra- 
tions.    Strongly  bound  in  leather,  raised  bands,  $7  50.    Cloth,  $6  50. 
MACLISE  (JOSEPH).     SURGICAL   ANATOMY.     In  one  large   im- 
perial quarto  vol.,  with  68  splendid  plates,  beautifully  colored;  con- 
taining 190  figures,  many  of  them  life  size.     Cloth,  $14. 
MEIGS  (CHAS.  D .).    ON  THE  NATURE,  SIGNS,  AND  TREATMENT 
OF  CHILDBED  FEVER.     In  one  Svo.  vol.  of  365  pages.     Cloth,  $2. 
MILLER  (JAMES) .    PRINCIPLES  OF  SURGERY.    Fourth  American, 
from  the  third  Edinburgh  edition.      In  one  large  8vo.  vol.  of  700 
pages,  with  240  illustrations.     Cloth,  $3  75. 

THE  PRACTICE  OF  SURGERY.     Fourth  American,  from  the 

last  Edinburgh  edition.  In  one  large  Svo.  vol.  of  700  pages,  with 
364  illustrations.  Cloth,  $3  75. 

MONTGOMERY  (W.  F.)  AN  EXPOSITION  OF  THE  SIGNS  AND 
SYMPTOMS  OF  PREGNANCY.  From  the  second  English  edition. 
In  one  handsome  Svo.  vol.  of  nearly  600  pages.  Cloth,  $3  75.  . 

MULLER  (J.)     PRINCIPLES  OF  PHYSICS  AND  METEOROLOGY. 
In  one   large  Svo.  vol.  with  550  wood-cuts,  and  two  colored  plates. 
Cloth,  $4  50. 
TUTIRABEAU;   A  LIFE  HISTORY.     In  one  12mo.  vol.     Cloth,  75  cts. 

MACFARLAND'S  TURKEY  AND  ITS  DESTINY.  In  2  TO!S.  royal 
12ino.  Cloth,  $2. 

M\RSH  (MRS.)  A  HISTORY  OF  THE  PROTESTANT  REFORMA- 
TION IN  FRANCE.  In  2  vols.  royal  12mo.  Cloth,  $2. 

ISJELIG  AN  (J.MOORE).  AN  ATLAS  OF  CUTANEOUS  DISEASES,  in 

JM     one  quarto  volume,  with  beautifully  colored  plates,  &c.  Cloth,  $5  50. 

NEILL  (JOHN)  AND  SMITH  (FRANCIS  G.)  COMPENDIUM  OF 
THE  VARIOUS  BRANCHES  OF  MEDICAL  SCIENCE.  In  one 
handsome  12mo.  vol.  of  about  1000  pnges,  with  374  wood-cuts. 
Cloth,  $4;  leather,  rui.sed  bands,  $4  75. 


HENRY  C.  LEA'S  PUBLICATIONS.  9 

NIEBUHR  (B.  G.)  LECTURES  ON  ANCIENT  HISTORY;  com- 
prising  the  history  of  the  Asiatic  Nations,  the  Egyptians, 
Greeks,  Macedonians,  and  Carthagenians.  Translated  by  Dr.  L. 
Schmitz.  In  three  neat  volumes,  crown  octavo.  Cloth,  $5  00. 

ODLING  (WILLIAM).  A  COURSE  OF  PRACTICAL  CHEMISTRY 
FOR  THE  USE  OF  MEDICAL  STUDENTS.  In  one  12mo.  vol. 
of  261  pp.,  with  75  illustrations.  Cloth,  $2. 

pLAYFAIR  (W.  S  )     A  TREATISE  ON  THE  SCIENCE  AND  PRAC- 
-L      TICE  OF  MIDWIFERY.     In  one  handsome  octavo  vol.  of  576  pp., 

with   166   illustrations,  and   two   plates.     Cloth,  $4 ;  leather,   $5. 

(Just  issued.} 

PAVY  (F.  W.)  A  TREATISE  ON  THE  FUNCTION  OF  DIGESTION, 
ITS  DISORDERS  AND  THEIR  TREATMENT.  From  the  second 
London  ed.  In  one  8vo.  vol.  of  246  pp.  Cloth,  $2. 

A  TREATISE  ON  FOOD  AND  DIETETICS,  PHYSIOLOGI- 
CALLY AND  THERAPEUTICALLY  CONSIDERED.  In  one  neat 
octavo  volume  of  about  500  pages.  Cloth,  $4  75.  (Jiist  issued.) 

pARRISH  (EDWARD).    A  TREATISE  ON  PHARMACY.    With  many 
•L      Formulae  and  Prescriptions.  Fourth  edition.  Enlarged  and  thoroughly 
revised  by  Thomas  S.  Wiegand.     In  one  handsome  8vo.  vol.  of  977 
pages,  with  280  illus.     Cloth,  $5  50  ;  leather,  $6  50. 

pIRRIE  (WILLIAM) .    THE  PRINCIPLES  AND  PRACTICE  OF  SUR- 
J-      GERY.     In  one  handsome  octavo  volume  of  780  pages,  with  316 
illustrations.     Cloth,  $3  75. 

pEREIRA  (JONATHAN).     MATERIA  MEDIC  A  AND  THERAPEU- 
•*-      TICS.     An  abridged  edition.     With  numerous  additions  and  refe- 
rences to  the  United  States  Pharmacopoeia.      By  Horatio  C.   Wood, 
M.  D.     In  one  large  octavo  volume,  of  1040  pages,  with  236  illustra- 
tions.    Cloth,  $7  00  5  leather,  raised  bands,  $8  00. 

pULSZKY'S  MEMOIRS  OF  AN  HUNGARIAN  LADY.    In   one  neat 
J-      royal  12mo.  vol.     Cloth,  $1. 

PAGET'S  HUNGARY  AND  TRANSYLVANIA.     In  two  royal  12mo. 
•L      vols.     Cloth,  $2. 

•DEMSEN  (IRA).     THE   PRINCIPLES    OF   CHEMISTRY.      In  one 
±l)     handsome  12mo.  vol      Cloth,  $1  50.      ((Just  issued.) 
pOBERTS  (WILLIAM).     A  PRACTICAL  TREATISE  ON  URINARY 
I«    AND  RENAL  DISEASES.     A  second  American,  from  the  second 
London  edition.     With  numerous  illustrations  and  a  colored  plate. 
In  one  very  handsome  8vo.  vol.  of  616  pages.     Cloth,  $4  50. 
•pAMSBOTHAM   (FRANCIS   H.)     THE   PRINCIPLES  AND    PRAC- 
Ib    TICE  OF  OBSTETRIC  MEDICINE  AND  SURGERY.     In  one  im- 
perial 8vo.  vol.  of  650  pages,  with  64  plates,  besides  numerous  wood- 
cuts in  the  text.     Strongly  bound  in  leather,  $7. 

EIGBY  (EDWARD).     A  SYSTEM  OF  MIDWIFERY.     Second  Ameri- 
can edition.    In  one  handsome  8vo.  vol.  of  422  pages.    Cloth,  $2  50. 
RANKE'S  HISTORY  OF  THE  TURKISH  AND  SPANISH  EMPIRES 
in  the  16th  and  beginning  of  17th  Century.     In  one  8vo.  volume, 
paper,  25  cts. 

HISTORY  OF  THE  REFORMATION  IN  GERMANY.     Parts  I., 

II.,  III.     In  one  vol.     Cloth,  $1. 

OCHAFER  (EDWARD  ALBER1).     A  COURSE  OF  PRACTICAL  HIS- 

W     TOLOGY  :  A  Manual  of  the  Microscope  for  M«dical  Students.     In 

one  handsome  octavo  vol.  With  many  illust.  Cloth,  $2.    (Just  Issued.) 


10  HENRY  C.  LEA'S  PUBLICATIONS. 

OMITH  (EUSTACE).  ON  THE  WASTING  DISEASES  OF  CHILDREN. 
^     Second  American  edition,  enlarged.     In  one  8vo.  vol.     Cloth,  $2  50. 

OARGENT  (F.  W.)     ON  BANDAGING  AND  OTHER  OPERATIONS 
*J     OF  MINOR  SURGERY.     New  edition,  with  an  additional  chapter 

on  Military  Surgery.     In  one  handsome  royal  12mo.  vol.  of  nearly 

400  pages,  with  184  wood-cuts.     Cloth,  $1  75. 

OMITH  (J.  LEWIS.)     A  TREATISE  ON  THE   DISEASES   OF   IN- 
^>    FANCY  AND  CHILDHOOD.     Third  Edition,  revised  and  enlarged. 

In  one  large  8vo.  volume  of  724  pages,  with  illustrations.     Cloth, 

$5;  leather,  $6.      (Just  issued.) 

OjHARPEY   (WILLIAM)    AND    QTJAIN   (JONES  AND   RICHARD). 
&     HUMAN  ANATOMY.     With  notes  and  additions  by  Jos.    Leidy, 

M.D.,  Prof,  of  Anatomy  in  the  University  of  Pennsylvania.     In  two 

large  8vo.  vols.  of  about  1300  pages,  with  51 1  illustrations.     Cloth,  $6. 

SXEY  (FREDERIC  C.)  OPERATIVE  SURGERY.  In  one  8vo.  vol. 
of  over  650  pages,  with  about  100  wood-cuts.  Cloth,  $3  25. 

SLADE  (D.  D.)  DIPHTHERIA  ;  ITS  NATURE  AND  TREATMENT. 
Second  edition.  In  one  neat  royal  12mo.  vol.  Cloth,  $1  25. 

SMITH  (HENRY  H.)  AND  HORNER  (WILLIAM  E.)  ANATOMICAL 
ATLAS.  Illustrative  of  the  structure  of  the  Human  Body.  In  one  large 
imperial  8vo.  vol.,  with  about  650  beautiful  figures.  Cloth,  $4  50. 

SMITH  (EDWARD).  CONSUMPTION ;  ITS  EARLY  AND  REME- 
DIABLE STAGES.  In  one  8vo.  vol.  of  254  pp.  Cloth,  $2  25. 

STILLE  (ALFRED).  THERAPEUTICS  AND  MATERIA  MEDIC  A. 
Fourth  edition,  revised  and  enlarged.  In  two  large  and  handsome 
volumes  8vo.  Cloth,  $10  ;  leather,  $12.  (Just  issued.} 

STILLE  (ALFRED)  AND  MAISCH  (JOHN  M.)  THE  NATIONAL 
DISPENSATORY:  Embracing  the  Chemistry,  Botany,  Materia 
Medica,  Pharmacy,  Pharmacodynamics,  and  Therapeutics  of  the 
Pharmacopoeias  of  the  United  States  and  Great  Britain.  For  the 
Use  of  Physicians  and  Pharmaceutists.  In  one  handsome  8vo.  vol  , 
with  numerous  illustrations,  (in press.) 

QCHMITZ  AND  ZTIMPT'S  CLASSICAL  SERIES.     In  royal  18mo. 
fc     CORNELII  NEPOTIS  LIBER  DE  EXCELLENTIBUS  DUCIBUS 

EXTERARUM  GENTIUM,  CUM  VITIS  CATONIS  ET  ATTICI. 

With  notes,  &c.     Price  in  cloth,  60  cents;  half  bound,  70  cts. 

C.  I.  CJESARIS  COMMENTARII  DE  BELLO  GALLICO.  With  notes, 
map,  and  other  illustrations.  Cloth,  60  cents;  half  bound,  70  cents. 

C.  C.  SALLUSTII  DE  BELLO  CATILINARIO  ET  JUGURTHINO. 
With  notes,  map,  &G.  Price  in  cloth,  60  cents  ;  half  bound,  70  cents. 

Q.  CURTII  RUFII  DE  GESTIS  ALEXANDRI  MAGNI  LIBRI  VIII. 
With  notes,  map,  &c.  Price  in  cloth,  80  cents  ;  half  bound,  90  cents. 

P.  VIRGILII  MARONIS   CARMINA   OMNIA.     Price  in  cloth,  85 

cents;  half  bound,  $1. 
M.  T.  CICERONIS  ORATIONES  SELECTEE  XII.     With  notes,  &c. 

Price  in  cloth,  70  cents  ;  half  bound,  80  cents. 

ECLOG^l  EX  Q.  HORATII  FLACCI  POEMATIBUS.  With  notes, 
Ac.  Price  in  cloth,  70  cents;  half  bound,  80  cents. 

ADVANCED  LATIN  EXERCISES,  WITH  SELECTIONS  FOR 
READING.  Revised.  Cloth,  price  60  cents  ;  half  bound,  70  cents. 


HENRY  C.  LEA'S  PUBLICATIONS.  11 


S WAYNE  (JOSEPH  GRIFFITHS).  OBSTETRIC  APHORISMS.  A 
new  American,  from  the  fifth  revised  English  edition.  With  addi- 
tions by  E.  R.  Hutchins,  M.  D.  In  one  small  12mo.  vol.  of  177  pp., 
with  illustrations.  Cloth,  $1  25. 

STURGES    (OCTAVITJS).     AN    INTRODUCTION    TO   THE   STUDY 
OF  CLINICAL   MEDICINE.     In  one  12mo.  vol.     Cloth,  $1   25. 
(NCHOEDLER  (FREDERICK)  AND  MEDLOCK  (HENRY).   WONDERS 
•^     OF  NATURE.   An  elementary  introduction  to  the  Sciences  of  Physics, 
Astronomy,     Chemistry,    Mineralogy,    Geology,    Botany,    Zoology, 
and  Physiology.     Translated  from  the  German  by  H.  Medlock.     In 
one  neat  8vo.  vol.,  with  679  illustrations.     Cloth,  $3. 

CjTOKES  (W.)     LECTURES  ON  FEVER.    In  one  8vo.  vol.    Cloth,  $2. 

SMALL  BOOKS  ON  GREAT  SUBJECTS.  Twelve  works ;  each  one  10 
cents,  sewed,  forming  a  neat  and  cheap  series  ;  or  done  up  in  3  vols., 
cloth,  $1  50. 

STRICKLAND    (AGNES).     LIVES  OF  THE  QUEENS  OF   HENRY 
>3     THE  VIII.  AND  OF  HIS  MOTHER.     In  one  crown  octavo  vol., 
extra  cloth,  $1;  black  cloth,  90  cents. 

MEMOIRS  OF  ELIZABETH,  SECOND  QUEEN  REGNANT  OF 

ENGLAND  AND  IRELAND.  In  onecrown  octavo  vol.,  extracloth, 
$140;  black  cloth,  $1  30. 

rpANNER  (THOMAS  HAWKES).    A  MANUAL  OF  CLINICAL  MEDI- 
-L     CINE  AND   PHYSICAL   DIAGNOSIS.     Third  American  from  the 
second  revised  English  edition.     Edited  by  Tilbury  Fox,  M.  D.     In 
one  handsome  12mo.  volume  of  366  pp.     Cloth,  $1  50. 

ON  THE   SIGNS  AND  DISEASES  OF  PREGNANCY.     From 

the  second  English  edition.  With  four  colored  plates  and  numerous 
illustrations  on  wood.  In  one  vol.  8vo.  of  about  500  pages.  Cloth, 
$4  25. 

rpUKE  (DANIEL  HACK).    INFLUENCE  OF  THE  MIND  UPON  THE 
•*•     BODY.     In  one  handsome  8vo.  vol.  of  416  pp.    Cloth,  $3  25. 
TiAYLOR    (ALFRED    S.)     MEDICAL    JURISPRUDENCE.     Seventh 
-•-     American  edition.     Edited  by  John  J.  Reese,  M.D.     In  one  large 
8vo.  volume  of  879  pages.     Cloth,  $5;  leather,  $6.     (Just  issued.) 
PRINCIPLES  AND  PRACTICE   OF    MEDICAL   JURISPRU- 
DENCE.    From  the  Second  English  Edition.      In  two  large  8vo. 
vols.     Cloth,  $10;  leather,  $12.      (Just  issued.} 

ON  POISONS  IN  RELATION  TO  MEDICINE  AND  MEDICAL 

JURISPRUDENCE.  Third  American  from  the  Third  London  Edi- 
tion. 1  vol.  8vo.  of  788  pages,  with  104  illustrations.  Cloth,  $5  50  ; 
leather,  $6  50.  ( Just  issued.) 

rpHOMAS  (T.  GAILLARD).    A   PRACTICAL  TREATISE  ON  THE 
J-     DISEASES  OF   FEMALES.     Fourth   edition,  thoroughly  revised. 
In  one  large  and  handsome  octavo  volume  of  801  pages,  with  ]91 
illustrations.     Cloth,  $5  00  ;  leather,  $6  00.     (Just  issued.) 
rpODD  (ROBERT  BENTLEY) .    CLINICAL  LECTURES  ON  CERTAIN 
J-     ACUTE  DISEASES.    In  one  vol.  8vo.  of  320  pp.,  cloth,  $2  50. 
rpHOMPSON  (SIR  HENRY).    CLINICAL  LECTURES  ON  DISEASES 
-L     OF  THE  URINARY  ORGANS.     Second  and  revised  edition.     In 
one  8vo.  volume,  with  illustrations.     Cloth,  $2  25.     (Just  issued.) 

THE  DISEASES  OF  THE  PROSTATE,  THEIR  PATHOLOGY 

AND  TREATMENT.  Fourth  edition,  revised.  In  one  very  h.-ind- 
some  8vo.  vol.  of  355  pp.,  with  13  plates.  Cloth,  $3  75. 


W 


12  HENRY  C.  LEA'S  PUBLICATIONS. 

rpHOMPSON  (SIR  HENRY).   THE  PATHOLOGY  AND  TREATMENT 
1     OF  STRICTURE  OF  THE  URETHRA  AND  URINARY  FISTULA. 

From  the  third  English  edition.     In  one  8vo.  vol.  of  359  pp.,  with 

illustrations.     Cloth,  $3  50. 

ALSHE  (W.  H.)  PRACTICAL  TREATISE  ON  THE  DISEASES 
OF  THE  HEART  AND  GREAT  VESSELS.  Third  American  from 
the  third  revised  London  edition.  In  one  8vo.  vol.  of  420  pages. 
Cloth,  $3. 

WATSON  (THOMAS).  LECTURES  ON  THE  PRINCIPLES  AND 
PRACTICE  OF  PHYSIC.  A  new  American  from  the  fifth  and  en- 
larged  English  edition,  with  additions  by  H.  Hartshorne,  M.D.  In 
two  large  and  handsome  octavo  volumes.  Cloth,  $9  ;  leather,  $11. 

WOHLER'S  OUTLINES  OF  ORGANIC  CHEMISTRY.  Translated 
from  the  8th  German  edition,  by  Ira  Remsen,  M.D.  In  one  neat 
12mo.  vol.  Cloth,  $3  00.  (Lately  issued.) 

(J.  SOELBERG).  A  TREATISE  ON  THE  DISEASES  OF 
THE  EYE.  Second  American,  from  the  Third  English  edition,  with 
additions  by  I.  Minis  Hays,  M.D.  In  one  large  and  handsome  octavo 
vol.,  with  6  colored  plates  and  many  wood-cuts,  also  selections  from 
the  test-types  of  Jaeger  and  Snellen.  Cloth,  $5  00  ;  leather,  $6  00. 

WHAT  TO  OBSERVE  AT  THE  BEDSIDE  AND  AFTER  DEATH 
IN  MEDICAL  CASES.  In  one  royal  12mo.  vol.  Cloth,  $1. 

WEST  (CHARLES).     LECTURES  ON  THE  DISEASES  PECULIAR 

W   TO  WOMEN.    Third  American  from  the  Third  English  edition.     In 

one  octavo  volume  of  550  pages.     Cloth,  $3  75  ;  leather,  $4  75. 

LECTURES  ON  THE  DISEASES  OF  INFANCY  AND  CHILD- 
HOOD.    Fifth  American  from  the  sixth  revised  English  edition.     In 
one  large  8vo.  vol.  of  670  closely  printed  pages.     Cloth,  $4  50  ;  lea- 
ther, $5  50.     (Just  issued.) 

ON   SOME    DISORDERS    OF    THE   NERVOUS   SYSTEM   IN 

CHILDHOOD.     From  the  London   Edition.     In  one   small  12mo. 
volume.     Cloth,  $1. 

WILLIAMS  (CHARLES  J.  B.  and  C.  T.)     PULMONARY  CONSUMP- 
VV  TION:    ITS   NATURE,  VARIETIES,    AND   TREATMENT.     In 

one  neat  octavo  volume.     Cloth,  $2  50. 

WILSON  (ERASMUS).  A  SYSTEM  OF  HUMAN  ANATOMY.  A 
new  and  revised  American  from  the  last  English  edition.  Illustrated 
with  397  engravings  on  wood.  In  one  handsome  8vo.  vol.  of  over 
600  pages.  Cloth,  $4  ;  leather,  $5. 

ON  DISEASES  OF  THE  SKIN.     The  seventh  American  from 

the  last  English  edition.     In  one  large  8vo.  vol.  of  over  800  pages. 
Cloth,  $5." 

Also,  A  SERIES  OF  PLATES,  illustrating  "Wilson  on  Diseases  of  the 
Skin,"  consisting  of  20  plates,  thirteen  of  which  are  beautifully 
colored,  representing  about  one  hundred  varieties  of  Disease.  $5  50. 

Also,  the  TEXT  AND  PLATES,  bound  in  one  volume.     Cloth,  $10. 

THE  STUDENT'S  BOOK  OF  CUTANEOUS  MEDICINE.     In 

one  handsome  royal  12mo.  vol.     Cloth,  $3  50. 

WINCKEL  ON   PATHOLOGY  AND  TREATMENT  OF  CHILDBED. 
With  Additions  by  the  Author.     Translated  by  Chadwick.     In  one 
handsome  octavo  volume  of  484  pages.     Cloth,  $4.     (Just  issued.) 
I7EISSL     ON    VENEREAL     DISEASES.       Translated     by    Sturgis. 
LI    (Preparing.) 


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